Delta
Delta function in leg morphogenesis (part 2/2) The expression of the Serrate and Delta genes patterns the
segments of the leg in Drosophila by a combination of their
signaling activities. Coincident stripes of Serrate and Delta
expressing cells activate Enhancer of split expression in
adjacent cells through Notch signaling. These cells form a
patterning boundary from which a putative secondary
signal leads to the development of leg joints. Elsewhere in
the tarsal segments, signaling by Dl and N is
necessary for the development of non-joint parts of the leg.
It is proposed that these two effects result from different
thresholds of N activation, which are translated into
different downstream gene expression effects. A general mechanism is proposed for creation of boundaries by Notch
signaling (Bishop, 1999).
The legs of Drosophila are jointed appendages, as in all other
arthropod animals. In Drosophila and most insects, the
structure of the legs is remarkably constant as follows:
every leg carries articulations, or joints, which divide the leg
into parts, or segments; these segments are called, from
proximal (or close to the body wall) to distal: coxa, trochanter,
femur, tibia and five tarsal segments, plus a pre-tarsus, or claw
organ, at the tip of the leg. The joints differ from
other parts of the leg in that they are devoid of bristles and
include a flexible intersegmental membrane and interlocking
parts composed of thickened cuticle. The details of the
morphology of these parts varies from joint to joint and only
the joints between the tarsal segments are identical and
composed of a 'socket' in the proximal part of the joint and an
interlocking 'ball' in the distal part. The other joints do not include a 'ball and socket'
structure but a variety of condyles and cavities (Bishop, 1999 and references).
Joints are not obvious in the developing legs until the time
of pupation, when the everting leg discs show a series of
constrictions, that different authors have identified with the
presumptive positions of the final joints. However, during subsequent
metamorphosis, the legs inflate and lose these constrictions
although different cell contours can still be seen at positions
that seem to correlate with future joints. The lack of markers other than morphological ones has
not allowed an exploration of joint development in more depth.
A marker that seems to correlate faithfully
with joints has been used, an enhancer trap inserted into the disconnected
(disco) gene. The disco gene encodes a
protein required for axonal migration and leg development
(Heilig, 1991) and in the legs, its expression is associated
with joint development. disco expression is present in the
developing leg imaginal disc at 120 hours after egg laying. Although it is expressed throughout the presumptive leg
region, disco expression is upgraded in a series of rings around
the center of the disc. During the first 4 hours after puparium
formation (APF), disco expression can be seen to become more
strongly modulated in rings, which correspond with the
presumptive leg segments. At around 12 hours APF,
lacZ expression is restricted to these rings, which in a lateral
view of the developing leg appear as stripes about 6 cells wide
situated next to but proximal to constrictions in the
presumptive tarsal region. Staining of adult flies
carrying this marker shows disco expression specifically
restricted to the joints (Bishop, 1999 and references).
Null alleles of Ser are lethal but a few mutant flies are formed
inside the pupal cases. These animals show a variety of
developmental defects, amongst them, leg deformities. The legs lack
joints between all segments although sometimes a remnant
constriction can still be seen. No other leg area is affected and
the segment boundaries are still present, as shown by the apical
bristles in tarsi and tibia. This mutant phenotype correlates
with the pattern of expression of Ser. Using an antibody against
the Ser protein, expression is seen to appear in rings in third
instar discs and later is
found close to the presumptive joint areas in pupal legs. Stripes
of Ser expression are seen proximal to constrictions in everting
legs. Double staining with disco expression shows
overlapping expression of disco and Ser.
However, in the bell-shaped distribution of disco, cells with
maximum levels of disco are located at the distal edge of the
stripe of Ser expressing cells.
These results show that Ser is required for the development
of joints, and Ser expression adjacent to joint areas suggests
that Ser could be directing joint development. If this were the
case it would be expected that ectopic expression of Ser would lead
to the ectopic development of joints, and indeed this
is the case. A klumpfuss-Gal4 line, which is expressed in the legs outside
the joints, was used to drive ectopic expression of a UAS-Ser
construct. klu-driven expression of Ser leads to the ectopic
development of joint-like cuticle, characterised by loss of
bristles, cuticle thickenings and inpocketings. In leg
regions like the tibia, where klu expression has defined
boundaries, ectopic constrictions tend to appear. The
transformation of interjoint leg regions towards joints is
corroborated by the accompanying ectopic expression of disco. Reciprocally, in Ser mutants, disco expression is lost
after 12 hours APF (Bishop, 1999 and references).
Dl expression has been revealed using a Dl-lacZ reporter allele
and an antibody against the Dl protein. Dl is
expressed throughout the presumptive leg at third instar, but
with upgraded expression in rings. In pupal legs these
rings can be seen to locate proximal to constrictions, and in
adult legs lacZ expression is found at low levels throughout the
leg, but it is stronger proximal to the joints. The expression of Ser, disco and Dl in pupal legs has been compared and the stripes of high Dl expression are found to coincide with cells
expressing Ser.
To study the requirements for Dl a viable
temperature-sensitive mutant combination of alleles was used that
produces mutant leg phenotypes. Following exposure to the restrictive temperature during
the third instar and pupal periods, when Dl is expressed near
the presumptive joints, the Dl mutant legs are shortened. The
tarsal segments are particularly reduced and have seemingly
disappeared, but on close inspection it can be seen that the
tarsal apical bristles are still present and sometimes some
remnant joint structures as well. Because Dl and Ser
are co-expressed, the joint defects in Dl mutants could be
indirectly due to a loss of Ser expression in Dl mutants. This was
examined and it was found that the Ser stripes are still present in
Dl mutant legs. Reciprocally, Dl expression is still
present in Ser mutants, showing that the
expression of Ser and Dl are not directly dependent on each
other and that their mutant joint phenotypes reflect independent
requirements. These results are interpreted as showing that
although joint areas require Dl for their development, the
strongest requirement for Dl is in the regions located between
segmental boundaries. This implies that the requirements for
Dl are more extensive than those of Ser, and this is
corroborated by their different requirements for disco
expression. In Ser mutant legs, the stripes of disco expression
form but are not maintained properly. However, in Dl mutants,
the stripes of disco are not formed correctly and instead wider
and fewer rings remain, a pattern similar to that of
Ser expression in Dl mutants. This disco
expression in Dl mutants is interpeted as a corroboration of the main
requirement for Dl being in the intersegmental regions. Failure
of these regions to develop causes the absence of non-disco
expressing cells between disco stripes so that fewer but wider
disco stripes appear in the mutants.
In spite of the differences in the requirements for Dl and Ser,
there is an overlap in function in that they are both required for the
development of joints. It is possible that this overlap explains
why the requirements of Dl in the joints are weaker than those
in the interjoints. It was decided to study whether this overlapping
requirement is mediated by N (Bishop, 1999).
N mutant flies
show a marked reduction in leg length with all areas of the leg
segments being affected. Joints are completely lost
but also often apical bristles. The overall length of the
segments, and especially of the tarsal region, is more reduced
than in Ser or Dl mutants because both joint and interjoint
tissue is missing. Thus, the N mutant phenotype looks
like a composition of the Ser and Dl mutant phenotypes. When
the expression of disco is revealed in N mutants, a combination
of Ser and Dl phenotypes is also seen. disco stripes do not
resolve properly, as in Dl mutants, and then they are
subsequently lost, as in Sermutants. Expressing a dominant-negative form of N in
the interjoint regions results in shortened legs, due to the loss of interjoint tissue,
but the joints are still present and sometimes fused.
These leg phenotypes thus resemble those produced in
interjoint regions by loss of Dl function.
Expression of a truncated and constitutively activated form
of N in the fourth tarsal segment
becoming hyper-jointed: double ball joints are formed. In addition, the interjoint region is reduced, either
as a consequence of its conversion to extra joint tissue, or to
an inability to develop the interjoint cell fates that have low,
but not high, levels of N activation.
Altogether these results suggest that the overlap of Ser and
Dl expression and requirements at the joints are mediated by N
activation. As a marker of N activity, the
expression of members of the E(spl) complex has been monitored. Using reporter constructs with the regulatory regions of
E(spl), which
reproduce the endogenous E(spl) expression in the leg discs, it can be seen that E(spl)m8 expression
is related to joints while m5 and presumably m6 are not.
Expression of the E(spl)m8 reporter construct in third instar
discs is initially strong in regions undergoing PNS
development. In the legs, these correspond to the chordotonal
organs in the femur and the tibia. In addition, expression near the presumptive joints is
seen to appear, and then resolve in the pupa into one-cell wide
stripes proximal to the leg constrictions, in positions
that correlate with cells with maximum levels of disco
expression. A similar although much weaker pattern of
expression of E(spl)mdelta is seen as revealed by the mAb323
antibody. Another marker
of N activity is the expression of N itself, which becomes
upregulated in cells where N signaling is being received. Using an anti-N antibody,
upregulated expression of N is seen immediately proximal to
constrictions in pupal legs. This upregulation is restricted to a single
row of cells at this position, thus confirming that
Ser and Dl are triggering N signaling in these cells (Bishop, 1999).
The results presented suggest a model in which the co-expression
of Ser and high levels of Dl in a stripe of cells
proximal to the future presumptive joints activate N in cells
adjacent but distal to this stripe. Could this
specificity be due to the presence of other factors that
would be interfering with Ser and Dl signaling in cells located
inside the Ser-Dl stripe or proximal to it? In the DV boundary
of the wing, the membrane protein encoded by the gene fringe
(fng) has been postulated to modulate N signaling by
interfering with Ser signaling. In the developing legs, fng is expressed
in stripes or rings around the positions of
presumptive joints. Using a UAS-fng
construct, fng was misexpressed.
Uniform tibial and tarsal fng expression only affects the joints, which are reduced
or disappear, a phenotype reminiscent of Ser
mutants. Thus, fng activity in the leg
seems to be restricted to a repression of joint
development around presumptive joint areas. It is
possible that fng expression in the wild type is
repressing N signaling in cells located in the Ser-Dl
stripe or proximal to it, providing the polarity in
the joint-promoting function of Ser and Dl (Bishop, 1999).
These results suggest a model in which the co-expression
of Ser and high levels of Dl in a stripe
of cells activate N in cells adjacent but distal to this
stripe. Activation of N promotes expression of members of the
E(spl) complex and leads to joint formation and disco
expression.
Loss of Dl eliminates first the regions between disco/Ser-expressing
rings, but also, secondly, joints. Since loss of
interjoint regions is also seen both in N mutants and following
expression of a dominant-negative form of N, it is postulated that
Dl expression in the interjoint regions produces low levels of
activation of N that do not lead to E(spl) expression but which
allow cell survival and/or cell proliferation.
Joint loss in Dl mutants is presumably
less severe than interjoint loss because Ser and Dl expression
could be synergistic and partially redundant. The combined and potentially
synergistic effects of Ser and Dl would produce a high level of
activation of N that would lead to expression of members of
the E(spl) complex, upregulation of N expression, and to joint
development and disco expression. Thus, it is believed that
combinations of signaling by Ser and Dl could produce
different levels of activation of N, which in turn are translated
into different downstream effects. As noted in other systems these downstream effects of N signaling
should be mediated by more factors than just E(spl), since
E(spl) mutant legs have been reported as having a wild-type
phenotype (Bishop, 1999 and references).
The width of the final joint region is wider than the single
row of cells activated by the membrane-tethered Ser and Dl
proteins and visualised by E(spl) expression. In principle it is
possible that the cells of the whole final joint all descend from
the E(spl) expressing cells, but previous studies have shown
that only one or two cell divisions occur in the legs after
puparium formation. Thus it is
likely that in the E(spl) expressing cells another cell signaling
molecule is activated, which in a secondary event would define
a wider joint presumptive region, just as N-induced expression
of the secreted signaling wingless protein defines the
presumptive wing margin. A reflection of this putative second signaling event in
the joints can be seen in the expression of disco. disco
expression is dependent on Ser but it is wider than the single
row of cells where N is activated and thus it cannot be directly
reflecting N signaling at the joint. However, the 'bell-shaped'
distribution of disco might reflect this putative secondary
signaling event, with a maximum in cells at the edge of the
Ser-Dl stripe. The nature of the joint-promoting putative
secondary signal is unknown at the moment, but one possible
component is the product of the four-jointed (fj) gene. The fj
protein is a putative signaling molecule that is expressed and
required at the joints. fj expression
has recently been shown to depend on fng and N signaling
during eye development, and it
is lost in N mutant legs (Bishop, 1999 and references).
An
autonomous negative effect of Dl and Ser does not explain why cells adjacent
but proximal to the Ser-Dl stripe do not seem to be signaled.
A possible explanation would be either an asymmetric
distribution of Ser and Dl, forming gradients like those seen
in the late third instar wing margin and
in ectopic expression situations, or a downregulation of N expression as has been
noted in the developing wing veins. The
Ser and Dl stripes in legs show no apparent asymmetry but
N distribution, although ubiquitous and initially uniform, becomes upregulated in cells distal to the Ser-Dl
stripes. Low availability of N protein could have an effect on
the intensity of N signaling, but since upregulation of N is in
itself a consequence of N signaling,
some other factor must polarize the signaling initially. Another
explanation would rely on the action of a repressor acting upon
cells proximal to the stripe. The phenotypes obtained after
ectopic expression of fng are consistent with such a role for
fng, as postulated in the wing. The expression of fng in the leg, which has been
described as complementary to that of E(spl), that is, present
in non-signaled cells but excluded from joint forming ones, is also consistent with this hypothesis. Such
a function of fng could also repress Ser and Dl signaling in
the stripe without recourse, or in addition, to putative
autonomous dominant negative effects of Ser and Dl. However,
other factors could also be involved, such as the cell polarity
pathway. Mutant phenotypes for dsh
and other members of the cell polarity pathway produce
ectopic joints with reversed polarity, which
appear just proximal to the position of Ser and Dl
stripes. Furthermore, in dsh mutants ectopic N
activation is seen proximal to the Ser-Dl stripe. Since the Dsh protein
has been shown to interact with N, and Dsh has been postulated
to inhibit N signaling in this manner,
the cell polarity pathway could be involved in repressing Ser
and Dl signaling to cells proximal to the Ser and Dl stripe (Bishop, 1999 and references).
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