Delta
Delta function in 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 (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).
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 (de Celis, 1998).
The possession of segmented appendages is a defining characteristic of the arthropods. By analyzing both loss-of-function and ectopic expression experiments, the Notch signaling pathway has been shown to play a fundamental role in the segmentation and growth of the Drosophila leg. Local activation of Notch is necessary and
sufficient to promote the formation of joints between segments. This segmentation process requires the participation of the Notch ligands, Serrate and Delta, as well
as Fringe. These three proteins are each expressed in the developing leg and antennal imaginal discs in a segmentally repeated pattern that is regulated downstream
of the action of Wingless and Decapentaplegic. While Dl expression overlaps fngand Ser, in some cases, it appears to extend into regions of the disc where neither fng nor Ser is expressed (Rauskolb, 1999).
fng mutant clones also result in fused joints and shortened legs. fng is required with the formation of all joints except the tibia-tarsal (ta1: basitarsus) joint. In most cases, the formation of the joints appears to be an autonomous property of wild type cells, while the failure to form joint structures is an autonomous property of cells mutant for Notch, Dl, Ser or fng. However, some exceptions have been observed in which joint formation is inhibited within wild type cells that border mutant clones or mutant cells appear to contribute to joint structure (Rauskolb, 1999).
The four-jointed (fj) gene encodes a type 2 transmembrane protein and is also expressed in concentric rings within the developing leg imaginal disc. In fj mutants, growth of the femur, tibia, and first three tarsal segments is reduced, and the ta2-ta3 segment border is absent. The rings of fj expression in leg imaginal discs are complementary to the rings of Nub expression. Consistent with this complementarity, fj expression is inhibited in cells expressing activated Notch; in cells neighboring ectopically expressing Ser or Dl, and in cells along the borders of ectopic fng expression. By contrast, fj expression is activated within cells expressing Ser or Dl. These observations indicate that fj is negatively regulated downstream of Notch signaling in the leg. Thus, Notch signaling subdivides each leg segment into distinct domains of gene expression (Rauskolb, 1999).
The Notch (N) signaling pathway is recruited for segregation of cell fates in a number of Drosophila tissue types. N dependent segmentation of Drosophila legs is regulated by a dynamic pattern of expression of its ligand, Delta (Dl). During third larval instar and early stages of pupation, high levels of Dl expression are seen in stripes of cells in the leg imaginal discs, which later form the
proximal borders of leg joints. These domains also display heightened Dl enhancer activity. During subsequent stages of pupation, following segmentation of the leg primordium, Dl expression becomes uniform throughout these segments barring the joints. Regulatory Dl mutations or mis-expression of Dl abolish leg segmentation. Domains of N signaling for segmentation of legs of flies are thus set up by a stringent spatial regulation of expression of the N ligand at the segment border. Further, a comparable role of Dl in antennal development reveals a common paradigm of Dl-N signaling for segmentation of appendages in flies (Mishra, 2001).
A enhancer trap line reported in this study reveals the domain of the developing leg segments where Dl enhancer activity is most intense, namely, the proximal borders of the presumptive leg joints. This interpretation is based on
the rationale that an overall weak detection of enhancer activity in this line limits its ability to mirror only the domains of highest Dl enhancer activity. Up-regulation of ligand expression is a pre-requisite for
activation of N signaling in neighboring cells. During wing development, for instance, a high level of Dl expression in the
presumptive vein forming regions induces N activity in the
intervein regions. Vein-intervein boundaries of wings are
thus set up by spatially regulated expression of the N ligand,
Dl. In the context of the leg segmentation too, an up-regulated Dl expression in the proximal borders of the leg joints provides a mechanism for segmentation due to N signaling in the adjoining groups of cells which acquire characteristic cell shapes to form the segmental constrictions. Loss of this characteristic pattern of Dl regulation, as in the case of its mis-expression induced by the ap-GAL4
driver, abolishes leg joint morphogenesis. Further,
leg phenotypes of recessive (Dlbl) and antimorphic alleles
(DlFE32) of Dl suggest, respectively, loss of its segmental regulation and overexpression. Both these conditions,
although producing opposing effects on Dl regulation,
display comparable leg phenotypes since in either case its
segmental modulation is compromised. Not surprisingly,
the leg phenotypes induced by Dl over-expression, and not those induced by a recessive loss (various Dlbl alleles) of its segmental regulation, are restored in a Nts background (Mishra, 2001).
N dependent segmentation of legs or the development of
vein-intervein boundaries in Drosophila wings have been
proposed to be developmental mechanisms designed to subdivide a morphogenetic field into smaller territories for local control of growth and patterning.
Indeed, these Dl mutant leg phenotypes provide strong
support for this view of leg segmentation where segment
boundaries serve as local sources for N signaling. Dl
mutant legs, lacking the segment boundaries, thus display
loss of both leg joints and growth. In summary,
these results show that one of the most critical elements in
N dependent leg segmentation is spatial regulation of
expressions of its ligands (Mishra, 2001).
The role of Dl during segmentation of legs is reminiscent
of that of its role in the development of vertebrate somites.
Also, in the latter, presumptive proximal and distal somite
borders display distinct levels of expression of the mammalian homologs of N ligand Dl. Given this strong conservation of the
N signaling pathway for the development of segment
boundaries, it is not surprising that segmentation of antenna
is also regulated by an identical mechanism. Leg-like distribution of the Dl-specific lacZ reporter has been noted when
antenna-to-leg transformations are induced by mutation in the homeotic gene Antennapedia. A common mechanism of Dl-N signaling for segmentation of antennae and legs thus operates with the caveat that it is spatially controlled by the hierarchy of regulatory pathways active in individual appendages. By extension, these results also imply that the diverse pattern of segmentation of appendages in different insect groups could be generated by spatial regulation of the N ligands. The range of variations in the number of segments in appendages of Dl mutants are reminiscent of
variations seen in not too distantly related dipteran groups
such as mosquitoes (e.g. 15 antennal segments) and horse fly
(seven antennal segments. Indeed, distinct sets of segment specific enhancers have been identified for the N ligand, Ser (Mishra, 2001).
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