Notch and leg morphogenesis (part 1/2) The legs of Drosophila are cylindrical appendages divided
into segments along the proximodistal axis by flexible structures called joints,
with each leg having 9 segments. The separation between segments is
already visible in the imaginal disc because folds of the epithelium and cells at
segment boundaries have a different morphology during
pupal development. The joints form at precise
positions along the proximodistal axis of the leg; both the
expression patterns of several genes in the leg and the results of
regeneration experiments suggest that different positions along
the proximodistal axis have different identities.
Two signaling molecules, wingless (wg) and decapentaplegic
(dpp) play a central role in patterning the leg discs. These genes
are activated in complementary anterior dorsal (dpp) and
anterior ventral (wg) sectors in response to the secreted protein
Hedgehog, which is only expressed in posterior cells. The asymmetry of
dpp and wg expression is maintained by mutual repression: dpp and wg
act antagonistically to regulate several genes involved in
generating differences along the dorsoventral axis. It is therefore likely
that the proximodistal patterning system initiated by wg and dpp determines the
localization of presumptive joints in developing leg discs, but
the identity of the gene products mediating this process is unknown (de Celis, 1998 and references).
Although the mechanism underlying joint formation is not
understood, the fusion of segments caused by some Notch
alleles indicates a requirement for Notch signaling.
In the leg imaginal disc most segments
form concentric rings, with the most distal in the center of the
disc. The exceptions are the
distal femur and proximal tibia, which are indistinguishable in
the larval imaginal disc and only separate during pupariation.
This separation occurs through the formation of lateral
invaginations that fuse creating two epithelial tubes constricted
at the femur/tibia joint.
When Notch activity is compromised in Nts1 larvae during
early and late third instar stage, the legs that develop are
misshapen, with some fusion between femur/tibia (early) and
tarsal (late) segments. To
determine more precisely where Notch activity is required
during leg development the phenotypes produced
by clones of cells that have greatly reduced Notch function were examined.
Two hypomorphic Notch alleles were used,
because unlike Notch null alleles they do not autonomously
prevent cell proliferation. In all the clones
of these Notch alleles that span a joint, no joint tissue is formed
by mutant cells. However, wild-type cells that are
in contact with Notch mutant cells are able to form joint
structures, so that incomplete joints form at normal locations
in mosaic joints. The effects of these two Notch mutations on
joint differentiation appears to be cell autonomous. In addition,
all clones in the anterior-ventral compartment
of the femur and tibia interfere with the
separation between the distal femur and the
proximal tibia. Legs carrying large
anterior or posterior Notch clones are always
shorter that their normal counterparts, and
mosaic tarsal segments have a 25% reduction
in area and a 30% reduction in length, as compared
with wild-type controls. Clones restricted to only one
leg segment do not affect the size of this
segment or the overall morphology and size of
the leg. Therefore it is only when clones span
the joint that both the defects in joint formation and the
global effects on the growth of the leg are seen (de Celis, 1998).
To distinguish which elements of the Notch
pathway are required during leg development,
clones of homozygous mutant
cells were generated, using lethal alleles in fng, Dl and Su(H)
as well as a deficiency of the E(spl) complex.
Lethal Ser alleles can survive into adults and
they have a low frequency of joint fusions.
The phenotype of Dl and Su(H) mosaics are
similar to each other and, like Notch, result in
a failure to make joints when mutant cells are
in the position where a joint should have
formed. Again, the wild-type
cells near the clones can still form joints, but
the length of the leg is reduced when the mutant
clones are large and span more than one
segment. In contrast, mutant cells homozygous
for a deficiency that removes the E(spl)bHLH
genes form normal joints even when they span
more than one segment and are characterized
by the differentiation of a vast array of ectopic
sensory organs. These develop
without intervening epidermal cells, indicating
that E(spl) is required for the lateral inhibition
mechanism that allows the spacing between sensory organs.
The larger clones cause a slight reduction in the overall size of
the leg (12% in area and 8% in length), but it is likely that these
effects are due to the differentiation of ectopic sensory organs
rather than direct effects on growth.
Cells mutant for fng also result in fusions between segments.
However, these effects are position dependent. Thus, with
clones spanning the boundary between the femur and tibia the
phenotypes are indistinguishable from those of Notch and
Su(H), resulting in a fusion of these two segments and
shortening of the leg, whereas in more distal
segments defects in the joint can only be detected between the
proximal two tarsal segments. The fact that fng is
important in leg segmentation suggests that boundaries similar
to the wing dorsal-ventral boundary are being created in at least
some of the presumptive joints (de Celis, 1998).
In the developing wing the localized activation of Notch can
be detected by the activation of certain target genes such as
E(spl) and vestigial. Furthermore, the domains of expression of Dl and Ser are
important in creating this localized activation of Notch. The expression of Ser,
Dl, fng, Notch and E(spl)m beta were therefore examined during leg development.
Heterogeneities in the expression of all these genes are
detected in the third instar imaginal disc, where Dl and
E(spl)m beta RNA are expressed in narrow concentric rings. In evaginating leg discs (0-4 hours APF) and in pupal legs, when the separation between leg segments becomes more
evident, E(spl)m beta expression is localized to a ring of distal cells
in each leg segment, suggesting that larval expression of E(spl)m beta also defines the distal end of each segment. The expression of fng is also restricted, and is only detected in
several broad rings localized to the presumptive tibia and first
tarsal segment, and in two groups of distal cells in the fifth
tarsal segment that could correspond to the presumptive claws. At this stage, no heterogeneity could be detected in the expression of Notch RNA, but by 24 hours
after puparium formation the levels are higher in the places
where the joints are being formed, which appear to
be the same cells where E(spl)m beta is expressed. At
these later stages, Dl also accumulates in rings of cells located
at the distal end of each segment and at the separation between
the femur and tibia, as well as in many clusters of cells that
correspond to developing sensory organs. Expression of E(spl) genes is dependent on Notch activity
and hence the localization of E(spl)m beta mRNA to rings of cells
in the imaginal and pupal leg disc indicates that there are high
levels of Notch activation in the distal-most set of cells in each
segment. To determine more precisely the relationship between
the E(spl)m beta-expressing cells and the expression of other
components of the Notch pathway, a reporter
gene was generated in which 1.5 kb of genomic DNA upstream of E(spl)m beta
was used to drive expression of a rat cell surface protein, CD2.
As a landmark for the segment boundaries an enhancer
trap in the bib gene, bib lacZ was used, which is expressed at higher levels
in single-cell wide rings at the distal end of each leg segment
during both larval and pupal development.
The expression of E(spl)m beta-CD2 is localized to a narrow
ring, 1-2 cells wide, which coincides with the cells expressing
bib lacZ and with cells that have higher levels of
lacZ expression in the N lacZ1 enhancer trap line.
The expression of N lacZ1 at the dorsoventral boundary and at
vein-intervein boundaries is dependent on Notch activity itself. Thus the coincident Notch,
E(spl)m beta and bib expression indicates that high levels of Notch
activation during imaginal leg development are restricted to the
most distal cells of each segment. The accumulation of Notch
ligands is also localized within the developing leg segments,
with the highest levels of Dl and Ser detected in a narrow stripe
of cells localized proximally to those expressing bib lacZ both
in the larval imaginal disc and at pupal stages (de Celis, 1998).
In agreement with the phenotypes obtained, expression of
fng, detected using a fng enhancer trap line, was found to be maximal in the tibia and first tarsal segment. Within these segments it appears
to be highest in the regions where bib lacZ and E(spl)m beta-CD2
are not expressed. Later, in evaginating discs, low
levels of fng-lacZ expression are also observed in distal tarsal
segments. During the formation of the wing
dorsoventral boundary fng is co-expressed with Ser, and appears to prevent Notch activation by Ser in dorsal
cells. It is likely that
Fng localization in the legs also contributes to the restriction of
Notch activity, and may be important in ensuring that activation
of Notch only occurs in the distal cells in the segment (de Celis, 1998).
The appearance of E(spl)m beta and bib expression in rings of cells should be an early indication of the subdivision of the leg disc into separate segments. Their expression develops progressively during the third larval instar, but because few molecular markers of individual segments have been described only a limited correspondence can be established between individual rings and leg segments in early stages of imaginal development. E(spl)mbeta-CD2 is first detected in second instar leg discs, before any indication of segmentation, in the most proximal cells of the leg epithelium. Later, in early third instar discs, a novel ring of E(spl)mbeta-CD2 develops in the centre of the disc, in the domain where the transcription factor Apterous is expressed. The expression of apterous is restricted to the cells of the fourth tarsal segment in late third instar and pupal discs. Therefore, it appears that the first segment boundary to be formed separates the presumptive tarsal segments 4 and 5. Subsequently, novel rings of both E(spl)m beta and bib expression develop close to this first central ring. Most of these are included within the domain of Distal-less expression, suggesting that they correspond to the developing boundaries between the tibia and t1 and between the tarsal segments t1 to t4. At later stages at least four tarsal segments can be identified by rings of E(spl)m beta and bib expression, and in addition novel domains of expression develop in the proximal region of the disc. These observations suggest that the boundaries between presumptive leg segments develop progressively. The first boundaries form in early-mid third instar larvae and correspond to the most distal segments. This temporal evolution is compatable with the observation that early leg discs forced to differentiate prematurely exclusively form structures that correspond to distal segments (de Celis, 1998).
To further characterize the relevance of restricted Notch activation during leg morphogenesis, the expression of several components of the Notch pathway was manipulated using the GAL4 system. In most of these experiments the driver line dpp-GAL4 was used: the dpp-GAL4 is expressed in an anterodorsal sector of leg discs with some weaker anteroventral expression also detectable. Expression of Necd, a dominant negative form of the Notch protein, using this driver line disrupts the joints between tarsal segments, leading to shortened legs. In contrast, expression of the intracellular domain of Notch (Ni), which has ligand-independent Notch activity, causes the formation of ectopic joint structures. The strength of these phenotypes depends on the UAS-Nintra line used. Thus, ectopic expression using dpp-GAL4 and a weak UAS-Nintra insertion (Nintra12.1) results in normal-size legs, which only develop partial joint-like structures, particularly in the tibia and first tarsal segment. The ectopic joints are incomplete, being restricted to the dorsal side of the leg where expression of GAL4 is highest. When a stronger UAS-Nintra line (UAS-Nintra79.2) was used in combination with the same driver, the legs are extremely abnormal in morphology and both the tibia and femur are bifurcated by an abnormal proximal-distal fold. The connections between tarsal segments are also affected, with many joint structures appearing in abnormal positions. These results demonstrate that Notch activity is both necessary and sufficient to trigger joint formation in leg cells. Overall the effects produced by ectopic Dl and Ser are similar: the altered morphology of the resulting legs includes both fusion of segments and ectopic joints. However there are positional differences in the way the ligands exert their effects. Thus, the strongest effects of mis-expressing Dl are observed in the tarsal segments, where joint formation is perturbed resulting in foreshortened fused tarsi. This resembles Notch loss-of-function phenotypes suggesting that the levels or position of Dl expression are interfering with normal Notch activity. In addition, an abnormal structure forms at the junction between the first and second tarsal segments, which seems to consist of a partial perpendicular joint. The strongest effects of Ser mis-expression are suggestive of dominant negative effects, since the tibia is foreshortened and forms abnormal joints with the femur and tarsi. In addition, incomplete ectopic joints can be observed at low frequency in distal tarsal segments. Thus, the phenotypes indicate that both activation and repression of Notch occurs when high levels of Notch ligands are expressed. It is likely that the differential effects of misexpression of Dl and Ser are related to the distribution of fng, because the strongest dominant negative effects of Ser occur in the tibia, where fng expression is maximal, and those of Dl occur in distal tarsal segments, where fng is absent or expressed at low levels. Similar effects occur when the ligands are expressed in the wing using the GAL4 system, where the outcome is in part determined by interactions between Notch and Fng. There is a good correlation between the adult phenotypes observed after mis-expression of Notch, Dl and Ser and the expression of bib lacZ , both in larval and pupal leg discs. Thus, ectopic expression of Necd always eliminates the dorsal (and occasionally the ventral) side of each ring of bib lacZ expression in all tarsal segments and Ni has the opposite effect, causing an extra dorsal stripe of bib lacZ expressing cells. In addition, ectopic expression of Ser leads to both activation and repression of bib lacZ . For example, novel proximal-distal stripes of bib lacZ expression are detected in the distal tarsal segments. The effects of Necd, Ni and Ser on bib lacZ expression are observed from the stage when bib lacZ expression is first detected, suggesting that Notch activity is required at the time when joint development between leg segments is initiated (de Celis, 1998).
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