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Genetic Control of Segmentation in Drosophila: Zygotic Gene Expression
by Dr. William Brook Department of Medical Biochemistry University
We previously discussed the maternally
localized factors that control segmentation in the Drosophila embryo. These factors influence the development of large portions
of the embryo: the anterior and posterior halves. During early embryogenesis,
these factors become distributed in concentration gradients. The nuclei
in the pre-cellular embryo read these gradients resulting in the subdivision
of the embryo into domains of gap gene expression. A generalization is that
gap gene expression patterns depend on activation by transcription factors
encoded by maternal and gap gene and is refined by repression by from other
gap gene transcription factors( A good review of this material can be found
in (Hoch and Jäckle, 1993; Kornberg and Tabata, 1993).
How is the maternal gradient information transformed
into gap gene expression patterns?
We have looked at hunchback already
hunchback activation by bicoid is an example of how a spatial
pattern of gene expression could be established by a concentration threshold.
(Remember : below a certain concentration bicoid would no longer
bind the promoter, so there would be no activation of hunchback in
We shall look at Krüppel as another
Krüppel is expressed as a stripe and so its regulation is more complicated
but can be summarized as follows:
- bicoid activates
activates Krüppel at low concentrations and represses Krüppel
at high concentrations
- knirps represses
What these genes do together is to define the expression
zone of Krüppel from both the anterior and the posterior sides.
Krüppel expression is activated by bicoid and hunchback
throughout most of its region. However, the expression of Krüppel
is repressed on the anterior and posterior sides by hunchback and
How is this studied at the molecular level? We shall look at the posterior
border of Krüppel expression, which is controlled by competition
between bicoid and knirps (Hoch et al., 1992).
Herbert Jäckle's group were studying the regulation of the gene Krüppel
and had identified a730 bp fragment that could drive the expression of a
lacZ reporter gene in the Krüppel pattern.
Using gel mobility shift assays, they found that
both bicoid and knirps bound to this region.
Furthermore, using DNase 1 footprinting they found that knirps
and bicoid bound to the same 16 bp region within the 730 bp
region. They made plasmid constructs that had seven copies of this 16-mer
in front of a CAT gene and transfected it into Drosophila tissue
They found that adding a plasmid expressing bicoid
increased the expression of CAT level in a dose dependent manner. When they
subsequently added a plasmid expressing knirps they found that they
could reduce the level of CAT expression. This suggests that bicoid
and knirps regulate the expression of Krüppel by competing
for binding at this 16bp site. When bicoid binds, Krüppel
is activated. When knirps binds, Krüppel is repressed.
Pair-rule genes (i.e. even-skipped, fushi tarazu)
The embryo is divided into large regions by the gap genes, which - along
with the co-ordinate genes - activate transcription of the pair-rule genes
in seven stripes of expression. The activation of the pair-rule genes in
striped patterns by the maternal coordinate genes and gap genes is the first
sign of segmentation in the embryo. Pair-rule gene expression patterns determine
the position of the parasegments.
How is this co ordinated? It turns out there is a hierarchy within the pair-rule
gene class. The primary pair-rule genes(even-skipped, hairy, and runt)
are activated directly by the gap genes and they regulate the expression
of the secondary pair-rule genes (fushi-tarazu, odd-skipped, odd-paired,
paired, and others) .
How are the primary pair-rule genes regulated? It turns out that there is
a separate enhancer controlling the expression of each stripe of gene expression
in each of the primary pair-rule genes. The first evidence for this came
from regulatory mutations that altered the transcription of specific stripes
of expression. This was shown conclusively when the regulatory regions were
analyzed using transgenes driving lacZ expression. It was discovered that
there were regulatory elements for each stripe of expression. So upstream
of even-skipped there are 7 independent regulatory elements - one
for each stripe. The three primary pair-rule genes are expressed in 7 stripes
each for a total of 21 over-lapping domains of gene expression. In principle,
the way each of these stripes of expression is established is no different
from the way the gap gene expression patterns are established through a
combination of positive and negative inputs.
Let's look at what is happening with the expression of even-skipped
in the second stripe from the anterior .
hunchback activate even-skipped stripe 2 expression. even-skipped
stripe 2 is repressed on the anterior side by the gap gene giant
and on the posterior side by Krüppel.(Small et al., 1991)
Figure modified from Small et al., 1991.
Upstream of the even-skipped gene is a 430
bp enhancer element which controls just the expression of even-skipped
in the stripe two region (it is aptly named the "stripe 2 enhancer"!).
This enhancer has 12 known factor binding sites, including 6 activator and
6 repressor sites. The 6 activator sites include 5 bicoid binding
sites and one hunchback site. There are 3 binding sites for each
of giant and Krüppel..
Diagram modified from (Arnosti et al., 1996)
regulation is an example of the complex regulation of spatial gene expression
by activation and repression at different control sites . As an example,
in the stripe 1 domain, the stripe 1 enhancer activates even-skipped transcription
and all others are inactive. In the domain between stripe 1 and two, all
seven enhancers are inactive. In the stripe 2 domain, the stripe 2 enhancer
activates transcription and all others are inactive, etc.
First phase of segment polarity gene expression:
pair-rule genes establish segment polarity gene expression patterns
The primary pair-rule gene expression patterns
are established by the coordinate and gap genes and then refined through
interactions with each other and with the secondary pair-rule genes. For
example, even-skipped (eve) represses the expression of
fushi-tarazu (ftz)leading the expression of the two genes in
complementary graded patterns of expression in alternating parasegments.
The pair-rule genes are already expressed in a periodic pattern, so it is
easy to imagine how they establish the segment polarity gene expression
in every parasegment. The expression patterns of the segment polarity genes
engrailed (en) and wingless (wg) are established through
positive and negative transcriptional regulation by the pair-rule genes.
For example, the expression of en is activated by either ftz
or eve in each parasegment, whereas wingless is repressed
by ftz or eve in each parasegment. (How would these interactions
be demonstrated, and how would it be shown that the interaction was direct
Other pair-rule genes also control wg and
en expression. For example, paired and odd-paired
are responsible for the activation of engrailed AND wingless in alternating
stripes. (N.B. this is different from what I told you in class.)
Parasegments and segments
These terms can be confusing. Parasegments and
segments are different ways of subdividing the cells along the a/p embryonic
axis . They are out of phase with each other. Parasegments correspond to
domains of gene expression and to an early morphological feature seen before
germ-band retraction, the parasegmental groove. At the time of the cellular
blastoderm, the cells within each segment are morphologically indistinguishable
from one another. The boundary between the parasegments is exactly between
the engrailed and wingless expressing cells and is marked
during gastrulation and early germ-band extension by a shallow groove. Beginning
after the completion of germ band extension and during germ band retraction,
the parasegmental grooves disappear at the same time as very deep grooves
begin to arise halfway down the length of each parasegment - these will
be the segmental boundaries.
Second phase of segment polarity gene expression:
cell to cell signaling
The regulation of the segment polarity genes by
the pair-rule genes is only the first stage of regulation. There are two
problems that must be overcome. First, the expression of the coordinate,
gap and pair-rule genes fade away and new mechanisms for regulating wingless
and engrailed are required. Furthermore, cellularization of the
embryo has occurred by this stage and it turns out that the mechanism for
maintaining wingless and engrailed expression are based on
cell to cell communication. This is why not all of the segment-polarity
genes are transcription factors.
How are the patterns of wingless and engrailed maintained?
Genetic experiments showed that en and wg are required for
each other's expression: wg disappears in en mutant embryos;
en disappears in wg embryos. This suggests that wg
and en maintain each other's expression. However, they are expressed
in completely different cells, so cell- cell communication must occur.
engrailed encodes a homeodomain protein; wingless encodes
a secreted peptide, a member of the WNT family. wingless is secreted
from the cells which make it. When the wingless protein binds to
its receptor on posterior cells, the signal is transduced to the nucleus
and maintains the transcription of engrailed.
A second signal must be invoked to explain the maintenance of wingless
expression by engrailed expression. Since engrailed
is a transcription factor, it cannot be directly responsible for the signal
from the posterior engrailed expressing cells. wingless and
engrailed expression are also lost in mutants for another
segment polarity gene is called hedgehog. hedgehog
is expressed in the same cells as engrailed and its expression is
lost in wingless and engrailed mutants. These results imply
that hedgehog is part of the genetic circuit linking wingless
and engrailed expression. hedgehog encodes a secreted factor
that is responsible for the signal from the engrailed expressing
cells back to the wingless expressing cells. hedgehog binds
to its receptor on the wingless expressing cells and this results
in the maintenance of wingless transcription.
One of the clues that hedgehog was likely
to be part of the circuit was that it had a phenotype that is very similar
to that of wingless. A series of different genes mutate to the same
phenotype (including cubitus interruptus, gooseberry, smoothened, fused,
armadillo, disheveled, porcupine), and most of these have turned out
to be part of either the wingless reception pathway or the hedgehog
Later in embryogenesis,wingless and engrailed expression become
independent of one another,but the spatial patterns remain the same. So,
the control of expression of the segment polarity genes wingless
and engrailed goes through several distinct phases.
How do segment polarity genes control pattern
in the segment?
The segment polarity genes control the pattern
of cell differentiation within each segment. It has been proposed by several
groups that wingless and hedgehog form concentration gradients
that act as morphogens specifying different fates within the segment much
in the same way that bicoid specifies fates within the anterior half
of the embryo. It is not yet clear that this is true but it is interesting
to think that mechanisms similar, in principle, to the ones used for patterning
the entire embryo, may also act to pattern each individual segment. (See
Peifer and Bejsovec, 1992, and DiNardo et al., 1994) for reviews
of the regulation of segment polarity genes.)
- The expression of the gap genes is regulated
by a combination of positive and negative transcriptional regulation mediated
by transcription factors encoded by the maternal coordinate genes and the
expression is controlled by bicoid, hunchback and knirps.
Competition between knirps and bicoid binding at the same
target sequence determines the posterior border of Krüppel
- The primary pair-rule genes are regulated directly
by the coordinate and gap genes each stripe of primary pair-rule gene expression
is regulated by a separate enhancer element.
- The pair-rule genes establish the periodic expression
patterns of wingless and engrailed expression
and engrailed maintain each other's expression as part of a regulatory
circuit involving cell-cell interactions.
- Outline the role of bicoid in the
regulation of the gap gene hunchback and in the regulation
of the gap gene Krüppel.
- There is a conflict between your text and the
results from (Hoch et al., 1992). The text says that bicoid
is a repressor of Krüppel but Hoch et al say
that it activates the expression of Krüppel. Explain
the conflict. (Hint: the claim that bicoid is a repressor
of Krüppel expression is based on the anterior expansion
of Krüppel transcription in embryos that lack bicoid
- Describe the types of experiments necessary to
make the diagram of the even-skipped stripe 2 enhancer shown above.
- Why must the segment polarity genes have more
than one mode of regulation.
See Peifer and Bejsovec, 1992, and DiNardo et
al., 1994) for reviews of the regulation of segment polarity genes.
Reviews* and References
Arnosti, D. N., Barolo, S., Levine, M., and Small,
S. (1996). The eve stripe 2 enhancer employs multiple modes of transcriptional
synergy. Development 122, 205-14.
*DiNardo, S., Heemskerk, J., Dougan, S., and O'Farrell, P. H. (1994). The
making of a maggot: patterning the Drosophila embryonic epidermis. Curr
Opin Genet Dev 4, 529-34.
Hoch, M., Gerwin, N., Taubert, H., and Jackle, H. (1992). Competition for
overlapping sites in the regulatory region of the Drosophila gene Kruppel.
Science 256, 94-7.
*Hoch, M., and Jackle, H. (1993). Transcriptional regulation and spatial
patterning in Drosophila. Curr Opin Genet Dev 3, 566-73.
*Kornberg, T. B., and Tabata, T. (1993). Segmentation of the Drosophila
embryo. Curr Opin Genet Dev 3, 585-94.
*Peifer, M., and Bejsovec, A. 1992. Knowing your neighbours: Cell interactions
determine intrasegmental patterning in Drosophila. Trends Genet. 8, 243-248.
Small, S., Kraut, R., Hoey, T., Warrior, R., and Levine, M. (1991). Transcriptional
regulation of a pair-rule stripe in Drosophila. Gene Develop 5, 827-839