Serrate
The Drosophila gene Serrate encodes a membrane spanning protein, which is expressed in a complex
pattern during embryogenesis and larval stages. Loss of Serrate function leads to larval lethality, which
is associated with several morphogenetic defects, including the failure to develop wings and halteres. It has been suggested that
Serrate acts as a short-range signal during wing development. It is required for
the induction of the organizing center at the dorsal/ventral compartment boundary, from which growth
and patterning of the wing is controlled. In order to understand the regulatory network required to
control the spatially and temporally dynamic expression of Serrate, its cis-regulatory
elements were analyzed by fusing various genomic fragments upstream of the reporter gene lacZ. Enhancer elements
reflecting the expression pattern of endogenous Serrate in embryonic and postembryonic tissues could
be confined to 26 kb of genomic DNA, including 9 kb of transcribed region. Expression in some
embryonic tissues is under the control of multiple enhancers located in the 5' region and in intron
sequences. The data presented here provide the tools to unravel the genetic network that regulates
Serrate during different developmental stages in diverse tissues (Bachmann, 1998a).
The product of the Drosophila gene Serrate acts as a short-range signal during wing development to
induce the organizing center at the dorsal/ventral compartment boundary, from which growth and
patterning of the wing is controlled. Regulatory elements reflecting the early Serrate expression in the
dorsal compartment of the wing disc have recently been confined to a genomic fragment in the
5'-upstream region of the gene (from -8 to -18 kb). This fragment, termed the dorsal wing regulator or DWR, responds to various
positive and negative inputs required for the early Serrate expression. Activation and maintenance
of expression in the dorsal compartment of the wing discs of second and early third instar larvae
depend on apterous, as revealed by reporter gene expression in discs either lacking or ectopically
expressing apterous. The DWR is not activated by ectopic fringe expression in the ventral compartment, suggesting that the observed induction of Serrate protein by ectopic Fringe is mediated by a different enhancer, which is active at later stages during wing development. The lack of Suppressor of Hairless results in a precocious repression of reporter gene expression along the margin, suggesting that the DWR of Ser responds to the postulated feedback loop mediated by the Notch signaling cascade to maintain expression in cells adjacent to the dorsal wing margin (Bachmann, 1998b).
Transcriptional downregulation during third larval instar is mediated by
hiiragi. hiiragi, which has not yet been cloned, develops a notched wing phenotype when homozygous and enhances the notched wing phenotype of SerD/+. Strikingly, in hirP1 homozygous third instar larvae the expression domain of the DWR not only persists on the dorsal wing pouch, but expands into the ventral compartment from mid-third instar onwards. hiiragi is a good candidate to be involved in the downregulation of the DWR of Ser. The lack of nubbin (nub) leads to the loss of wing structures. In discs mutant for nub expression, the DWR along the D/V boundary is upregulated and persists longer than in wild-type discs. This is in agreement with the observation that Serrate protein expression appears to be more pronounced along the dorsal wing margin in nubbin mutant discs. This regulatory element also responds to Delta signaling in a nonautonomous way to maintain
Serrate expression along the dorsal margin. The results clearly show that some of the previously
described transactivators of Serrate protein expression, e.g. fringe, act on elements required for later
aspects of Serrate expression (Bachmann, 1998b).
Drosophila wing development is a useful model to study organogenesis, which requires the input of selector genes that specify the identity of various morphogenetic fields and cell signaling molecules. In order to understand how the integration of multiple signaling pathways and selector proteins can be achieved during wing development, the regulatory network that controls the expression of Serrate (Ser), a ligand for the Notch (N) signaling pathway, which is essential for the development of the Drosophila wing, as well as vertebrate limbs, was examined. A 794 bp cis-regulatory element located in the 3' region of the Ser gene can recapitulate the dynamic patterns of endogenous Ser expression during wing development. Using this enhancer element, Apterous (Ap, a selector protein), and the Notch and Wingless (Wg) signaling pathways, are shown to sequentially control wing development through direct regulation of Ser expression in early, mid and late third instar stages, respectively. In addition, later Ser expression in the presumptive vein cells is controlled by the Egfr pathway. Thus, a cis-regulatory element is sequentially regulated by multiple signaling pathways and a selector protein during Drosophila wing development. Such a mechanism is possibly conserved in the appendage outgrowth of other arthropods and vertebrates (Yan, 2004).
Ser is expressed in the dorsal compartment during the early stages
of wing disc development. This expression pattern is identical to that of the selector gene of the dorsal compartment, ap, which encodes a homeodomain
transcription factor. It has been hypothesized that early Ser expression
in the dorsal compartment is under the direct control of Ap.
However, no direct evidence has been shown to support this hypothesis. To
determine whether Ser is a direct target gene of Ap,
whether the 794 bp Ser minimal wing enhancer is regulated by Ap was tested. Construct 10, Ser-lacZ containing
the 794 bp Ser minimal enhancer, is expressed in a stripe in the
dorsal compartment flanking the DV boundary at 24 hours after the L2/L3 molt
in early third instar. A constitutively active form of Ap (ChAp) was expressed using the Gal4/UAS system and Ser-lacZ expression was examined. When
Dpp-Gal4 was used to drive ChAp expression at the anteroposterior (AP) boundary, ectopic Ser-lacZ expression was found in the ventral wing regions along
the AP boundary, overlapping dpp-Gal4 expression in early and late third
instar. This
indicated that Ap is sufficient to activate Ser expression, probably
cell-autonomously. To determine whether Ap function is necessary for
Ser expression, an Ap antagonist, dLMO, was expressed in
cells along the AP boundary, using a patched (ptc) promoter. This led
to the loss of Ser-lacZ expression in the early third instar and
partial reduction of Ser-lacZ in the late third instar, suggesting
that Ap is required in vivo for Ser expression in the dorsal
compartment (Yan, 2004).
To test whether early Ser expression can be directly regulated by
Ap, DNaseI footprinting analysis was used to determine the interaction sites
between the 794 bp DNA sequence and Ap. A total of 14 protected Ap binding
sites were detected spanning the 794 bp element. The binding of Ap to
this Ser minimal wing enhancer is sequence specific with two major
binding sequences, TAATNN and CAATNN. The TAATNN consensus sequence matches the six-nucleotide
consensus binding sequence for homeodomain proteins. There
is also the non-canonical CAATNN consensus sequence derived from the aligned
sequences, which matches the consensus binding sites for some homeodomain
proteins, such as murine S8. The existence of four CAATNN sites suggests that Ap
may bind the CAATNN sequences specifically, in addition to the canonical
TAATNN sites (Yan, 2004).
To test whether these Ap binding sites are functionally important in vivo,
nucleotides in the Ap-binding sequences of Ser-lacZ
construct 10 were mutagenized from TAATNN and CAATNN to AAAANN or TTTTNN, in most cases. The
(mAp)Ser-lacZ construct, which included mutations in all 14
Ap-binding sites, showed no enhancer activity in the wing and haltere discs in
early third instar, as compared with Ser-lacZ expression, which was
first detected in much of the dorsal compartment and then as a dorsal stripe. In mid and late third instar, (mAp)Ser-lacZ expression was reduced or eliminated. These results
show that the Ap-binding sites identified in vitro are crucial for the
activity of the 794 bp Ser minimal wing enhancer in vivo. In summary,
Ser expression is mediated by direct Ap interaction with the 794 bp
wing enhancer during the early third instar stage were mutagenized (Yan, 2004).
The results reported here demonstrate that a 794 bp cis-acting regulatory
module in the Ser locus can be temporally regulated by three distinct
mechanisms that are employed for the proper establishment of the DV organizer
during wing development. (1) The selector protein Ap directly activates
Ser expression in the dorsal compartment during the early third
instar, which sets up N activation for the next stage. (2) By the middle
of the third instar, the N pathway maintains Ser expression by a
positive-feedback loop along the DV boundary. This feedback loop maintains Ser
and Dl expression, leading to the activation of N signaling at the DV
boundary, which is essential for establishing the DV organizer. (3) At the end of the third instar, as a result of Wg signaling, Ser is
expressed in two stripes flanking the DV boundary, which limits N activation
to the DV border. In
addition, Ser expression in provein cells
is dependent on input from the Egfr pathway. These results indicate how
tissue-specific selector and signaling molecules can work sequentially to
achieve a complex developmental process, such as organogenesis, which involves
a complex temporal and spatial regulation of genes. However, the conclusion
that the Ser minimal wing enhancer is sequentially regulated by Ap,
Notch, Wg and Egfr does not exclude the possibility that these
molecules/signaling pathways may cooperate and synergistically stimulate gene
expression at certain stages. In this case, mutations that specifically impair
response to the intended factor would affect Ser-lacZ expression in
other phases of disc development (Yan, 2004).
Evidence is provided that Ser is indeed a direct target gene
for Ap, thus forming a link between Ap, which specifies dorsal identity, and
the signaling pathways that organize the DV boundary. Ap may regulate the
Drosophila FMRFa neuropeptide gene and a mouse glycoprotein hormone
alpha-subunit gene enhancer by binding to TAATNN sequences. Thus,
TAATNN sequences may regulate most, if not all, Ap target genes (Yan, 2004).
The 794 bp Ser minimal wing enhancer is regulated by Ap, and is
expressed in the dorsal compartment of wing and haltere discs at 7.5 hours
after the L2/L3 molt in early third instar. The 7.4 kb
5' flanking sequence and the 8 kb 3' flanking sequence can also direct reporter gene expression in all of the dorsal compartment during wing development. A
9.5 kb Ap cis-response element was also isolated ~7.5 kb upstream of the
Ser translational initiation site,
although it is not clear whether Ap directly regulates this element. Further
dissection of this element into smaller fragments did not succeed in
recapitulating the dorsal anlage expression pattern.
These results suggest that crosstalk between different cis-elements is
required to regulate Ser dorsal expression, and that there is more
than one Ap response element at the Ser locus.
Given the importance of Ap-regulated Ser expression, multiple Ap
response elements might be expected. Enhancer redundancy has been observed in
many genes and may have evolved as a protection against loss of gene activity
when mutations occur in regulatory sequences (Yan, 2004).
Around 24 hours after the L2/L3 molt, a transition occurs in Ser
minimal enhancer expression from all dorsal cells to dorsal cells near the DV
boundary [24 hours after the L2/L3 molt is defined as early third instar
because 48-72 hours AEL (after egg laying) is generally taken as the early
third instar, which is equal to 0-24 h after the L2/L3 molt]. During this
transition, Ser expression in dorsal cells flanking the DV boundary
may be regulated by Ap, as well as by the N pathway. At 24
hours after the L2/L3 molt, (mAp)Ser-lacZ displays no activity, and
[mSu(H)]Ser-lacZ expression is evident in dorsal cells near the DV
boundary. Although these data suggest that Ap regulates Ser expression
in dorsal cells near the DV boundary, they do not exclude the possibility that
Notch may still be involved in directly regulating Ser expression
during this transition, since Su(H) may still be able to bind to and activate
[mSu(H)]Ser-lacZ (Yan, 2004).
Activation of N signaling at the nascent DV boundary is essential for the
formation of the DV boundary. Ser and Dl are highly expressed at the DV border in
mid-third instar and their expression can be ectopically activated by a
constitutively active form of N, which suggests a positive-feedback loop
between N ligands and the receptor. The activation of such a feedback loop between N and
its ligands is likely to be among the earliest events in the formation of the
DV boundary. The finding that the Ser wing enhancer is regulated by
the N pathway, and that two Su(H)-binding sites are required for the in vivo
activity of this enhancer in the mid third instar, suggests that N signaling
can directly regulate Ser expression through Su(H). Although these
results are consistent with direct activation of the Ser gene by
Su(H), they do not preclude the possibility that N signaling may regulate
Ser through other transcription factors, possibly downstream of
Su(H). This would explain why [mSu(H)]Ser-lacZ showed a significant,
but not dramatic, loss of enhancer activity. Alternatively, it
remains possible that Su(H) can still bind to and activate at least one of the
two mutant Su(H) binding sites in [mSu(H)]Ser-lacZ (Yan, 2004).
In vitro and in vivo results suggest that the regulation of
Ser by Wg signaling occurs directly through dTCF. Using DNase I
footprinting, two major classes of dTCF binding sequences were found: the dTCF
consensus sequence CCTTTGATCTT and the HMG consensus sequence WTTGWW, which
are consistent with previously identified dTCF binding sequences.
Interestingly, the presence of dTCF/HMG binding sites in the Ser
minimal wing enhancer may explain the crosstalk observed between the 3'
Ser enhancer and the 5' Ser promoter. HMG proteins can bend
DNA, and could therefore bring the 3' enhancer close enough to interact
with the transcriptional machinery binding at the 5' promoter (Yan, 2004).
In late third instar, Wg signaling is maintained in the DV organizer by the
N pathway. Wg signaling activates Ser and Dl
expression in the cells flanking the DV boundary, which in turn activates N
signaling to maintain a positive-feedback loop between N and Wg signals.
Because of an autonomous repression effect of N ligands on their receptor,
Ser and Dl expression in the flanking cells also prevents N
signaling from spreading out of the DV border. N signaling then turns off
Ser and Dl expression by inducing cut at the border.
Although the molecular nature of the dominant-negative effects of N ligands,
and the repression of Ser and Dl by N signaling remains unknown, these
mechanisms may play important roles in keeping the boundary sharp.
Interestingly, the Ser minimal wing enhancer is also repressed at the
DV border, suggesting that it is possible to study the molecular mechanism of
Ser repression at the border using this 794 bp enhancer (Yan, 2004).
Ser is expressed in provein cells and its expression is regulated by Egfr signaling at the pupal stage. N signaling also plays an important role in determining vein cell fate. The data on Ser expression in provein cells is consistent with a report on Ser function during vein development. Thus, in
addition to its essential role in development of the Drosophila leg
and vertebrate limbs, Egfr/Fgf signaling also plays a role in
Drosophila wing development, suggesting a conserved role of Egfr
signaling in 'appendage' development. Ser expression was examined in
both gain-of-function (gof) and loss-of-function (lof) Egfr signaling-mutant
backgrounds. First, in a rho gof mutant
(UAS-rho*), Ser appeared to be
ectopically expressed between L3 and L4, exactly where
ectopic rho activity was localized. Ser expression in the proveins was eliminated in a rho and vein (vn, encoding a Egfr ligand) double-mutant (Egfr lof) background, in which vein formation is completely abolished. These results suggest that the Egfr pathway may regulate Ser expression during vein development at the pupal stage. Interestingly, the Ser minimal wing enhancer is expressed in provein cells at both larval and pupal stages. Further investigation of this element may shed light
on how Egfr signaling regulates vein differentiation (Yan, 2004).
Given that the Ser-Fng-N pathway is evolutionarily conserved in appendage
development between insects and vertebrates, the mechanism by which Ser is sequentially
regulated by Ap, N, Wg and Egfr may also be conserved in appendage outgrowth
of other arthropods and vertebrates. Consistent with this hypothesis, the Ap,
Wg/Wnt and Egfr/Fgf pathways are also involved in appendage development in
vertebrates, as well as D. melanogaster. Indeed,
a BLAST search of the Drosophila pseudoobscura genome identified a
putative homolog of the Ser minimal wing enhancer. Interestingly,
this enhancer region is also located less than 1 kb downstream of the putative
D. pseudoobscura Ser 3'UTR. Sequence comparisons between the
Ser minimal wing enhancer from D. melanogaster and the
putative D. pseudoobscura enhancer show a significant degree of
similarity, whereas the similarities in the 5' and 3' flanking
regions are lower. Importantly, sequences of putative Ap, Su(H) and dTCF binding sites are highly conserved in D. pseudoobscura and D. melanogaster. Although the strong conservation of sequence and location suggests that the putative D. pseudoobscura Ser enhancer may be a functional homolog of the
D. melanogaster Ser minimal wing enhancer, it remains to be tested
whether this enhancer drives reporter gene expression at the identical time
and location in the D. melanogaster wing discs (Yan, 2004).
Spitz, and spitz group genes are at the top of the regulatory hierarchy in the development of salivary ducts. The salivary primordium consists of two regions, a more dorsal pregland anlage and a ventral preduct anlage. Spitz signaling to ventral cells, through the EGF-receptor acts to block forkhead expression in preduct cells, thereby restricting gland identity to more dorsal cells. Forkhead acts in dorsal pregland cells to block duct fate, specifically acting to repress Serrate, a duct specific gene as well as breathless and trachealess, also required for duct formation. The spitz group genes rhomboid and pointed are required for duct fate (Kuo, 1996).
Serrate is induced in the wing disc wherever cells expressing fringe are juxtaposed to cells not expressing fringe. Fringe is a secreted protein that targets Serrate by an unknown mechanism. Thus Serrate is found in the dorsal compartment of the wing imaginal disc (Kim, 1995).
Serrate may be a target of Apterous. The way Apterous works appears to be complex, having at least two distinct outputs. First, Apterous is responsible for making the dorsal cell distinct from ventral, a property that may be due to its activating the gene Dorsal wing. Second, Apterous regulates the expression of boundary determining proteins such as Fringe and Serrate. fringe, regulated by Apterous, targets Serrate by an apterous independent mechanism (Kim, 1995). Serrate appears to signal from dorsal to ventral cells to elicit the production of a long range morphogen, perhaps Wingless. In this, Serrate functions through Notch. Fringe is remarkable because a boundary forms wherever fringe-expressing and nonexpressing cells meet, a boundary that can organize long-range pattern (Lawrence, 1996).
Notch function is required at the dorsoventral boundary of the developing Drosophila wing for its normal growth and patterning. Clones of cells expressing either Notch or its ligands Delta and Serrate in the wing mimic Notch activation at the dorsoventral
boundary, producing non-autonomous effects on proliferation and activating expression of the target genes E(spl), wingless and cut.
The analysis of these clones reveals several mechanisms important for maintaining and delimiting Notch function at the dorsoventral
boundary: The secreted proteins Wingless and Hedgehog are essential
to the elaboration of the denticle pattern in the epidermis
of Drosophila embryos. Signaling by Wingless
and Hedgehog regulates the expression of veinlet
(rhomboid) and Serrate, two genes expressed in prospective
denticle belts. Thus, Serrate and veinlet (rhom) partake in
the last layer of the segmentation cascade. Ultimately,
Wingless, Hedgehog, Veinlet (an indirect activator of the
Egfr) and Serrate (an activator of Notch) are expressed in
non-overlapping narrow stripes. The interface between any
two stripes allows a reliable prediction of individual
denticle types and polarity, suggesting that contact-dependent
signaling modulates individual cell fates.
Attributes of a morphogen can be ascribed to Hedgehog in
this system. However, no single morphogen organizes the
whole denticle pattern (Alexandre, 1999).
Both Wingless and Hedgehog signaling pathways repress Serrate
expression. Since both pathways are believed to activate
transcription, it is imagined that they activate the expression of a
repressor of Serrate. In addition, Serrate may also be negatively
regulated by the transcriptional repressor Engrailed. In contrast
to Serrate, veinlet is regulated both positively and
negatively: it is repressed by Wingless and activated by Hedgehog.
In addition to this vertical flow of information, regulatory
interactions also exist between veinlet and Serrate. At
the least, Serrate activates veinlet expression by way
of the Notch pathway. This effect is purely non-cell
autonomous. In contrast, Serrate appears to repress veinlet in a cell autonomous manner (indeed, in cells where
it is expressed, Serrate represses the Notch pathway). However, it is also possible that
whichever mechanism activates Serrate expression also
represses veinlet expression. This would explain why
the expression of Serrate and veinlet is always
mutually exclusive (Alexandre, 1999).
The regulatory interactions summarized above are sufficient
to explain the spatial pattern of both Serrate and
veinlet expression. Non-autonomous
repression of Serrate by Wingless and Hedgehog ensures that
Serrate is expressed in stripes. The spread
of Wingless toward the anterior defines the posterior edge of
the domain of Serrate expression. Similarly, the anterior edge
of the Serrate domain appears to be specified over three cell
diameters by Hedgehog slightly further than expected
since Hedgehog is thought to act only over 1-2 cells in
Drosophila embryos. Expression of veinlet is activated by two different signals, Hedgehog at the
anterior and Serrate at the posterior. Although Hedgehog
signaling is symmetrical, it does not activate veinlet (rhom)
expression anteriorly because
there, Wingless represses veinlet expression. Likewise,
Serrate activates veinlet expression but only on one side
because of unilateral repression by Wingless (Alexandre, 1999).
These interactions display a clear temporal hierarchy. The
secreted molecules Hedgehog and Wingless are expressed first
and where they do not reach, Serrate expression is
subsequently allowed. At stage 11, Hedgehog and Serrate
activates veinlet expression in separate cells.
Ultimately, this chain of interactions results in detailed patterns
of gene expression (Alexandre, 1999).
Mapping the expression pattern of various genes onto the
denticle pattern suggests simple correlations.
These correlations have allowed the visualization of pattern where it
was previously thought there was none, as in wingless mutants. It is now believed that wingless mutants make denticle
type 3, 4 and 5 and not exclusively type 5 as has been suggested. The correlations provide a
guide to understand various phenotypes such as those of
patched mutants and wg-en-double mutants. In wg-en-double
mutants, the correlation between gene expression and denticle
type/polarity is particularly evident. Expression of veinlet is in circles surrounded by Serrate
expression; this correlates with polarity reversal in the
cuticle. Non-uniform gene expression shows that these
embryos have more pattern than previously noted. For such embryos to be truly unpatterned, they
would have to express Serrate uniformly as well as not express
veinlet (rhom). This may occur in wg-en-hh- triple mutants
since they may not contain any repressor of Serrate. It is
presumed that the converse situation (Serrate 'off'and veinlet
'on' everywhere) would also lead to unpatterned
embryos. This situation would prevail in wg-ptc-en-triple mutants.
Although the correlations have good predictive value, they suffer from several limitations. (1) Denticle shape
does not necessarily reflect an integer value. Indeed,
unambiguous typing is not always possible and exact denticle
shapes vary from segment to segment. (2) Causal
relationships between the activation of a particular signaling
pathway and a given denticle type still remain to be
investigated. The various signaling pathways are predicted to
control cytoskeletal behavior, which in turn affects denticle
shape and cell polarity. Local polarity reversals indicate that
individual cells are able to locate the source of a particular
signal, suggesting that subcellular signaling complexes control
the cytoskeleton directly. (3) The
involvement of additional regulators cannot be excluded. In particular, it is possible
that redundant regulators of the Notch and Egfr pathway
contribute to the choice of denticle type. These could include
Vein (another Egfr ligand), Delta (a
Notch ligand) or possibly Fringe. vein is
not required for embryogenesis
suggesting that it does not play an important role if any.
Possible contributions from Delta to denticle patterning are not
readily assessed because of Deltas earlier action in
neurogenesis (Alexandre, 1999).
These results show that no single morphogen organizes the
denticle pattern: patterning arises, at least initially, from the
combined actions of Wingless and Hedgehog.
Wingless is clearly not involved in the specification of
denticle types (or diversity) across each belt since it does not
act in this region of the epidermis. If it did, veinlet and
Serrate would not be expressed because, as has been shown,
they are both repressed by Wingless. Nevertheless, Wingless
acts at a distance, over 3- to 5-cell diameters to set the
boundaries of the Serrate expression domain and thus
establishes conditions for subsequent juxtacrine signaling.
Long-range Wingless action is also required for the
asymmetric action of Serrate: Serrate does not activate veinlet
(rhom) expression posteriorly because of the presence of
Wingless there, 3- to 5-cell diameters from the site of wingless
transcription. In this sense, Wingless modulates, at a distance,
the outcome of local signaling. In neither of these activities is
there evidence for concentration-dependent signaling.
However, one cannot formally exclude the possibility that the
specification of type 6 denticle requires low-level Wingless.
Furthermore, the suggestion that Wingless is not a morphogen
in the embryonic epidermis is at odds with studies of the first
thoracic segment where various levels of Wingless signaling
lead to the specification of distinct cuticular structures. Re-assessment of these phenotypes
with early molecular markers might tell whether or not
Wingless acts directly in a concentration-dependent manner in
the embryonic epidermis (Alexandre, 1999).
The situation with Hedgehog is clearer since it has
qualitatively distinct effects over a narrow strip of cells. It activates veinlet expression in adjoining
posterior cells while its repressive effect on Serrate expression
extends over three cell diameters. This suggests that, at high
level, Hedgehog activates veinlet (near the source)
while at both low and high levels it repress Serrate expression
(further away from the source). In this sense, Hedgehog
qualifies as a morphogen. Whether differential
responses at different distances from the Hedgehog source
reflect true concentration dependence remains to be assessed.
It is noted that the repressive effect of Hedgehog on Serrate
expression might take place early in development since, in
wingless mutants, hedgehog expression decays around stage 10 and yet Serrate expression is still confined
at the anterior. It is suggested that early Hedgehog has
a repressive effect on Serrate expression that lasts at least until
stage 11, when veinlet expression commences. It is
therefore conceivable that the 3-cell-wide domain where
Serrate is repressed at stage 11 originates by cell proliferation
from a single row of cells that abut the Hedgehog source at
early embryonic stages. According to this scenario, the effects
of Hedgehog on Serrate and veinlet expression would
both be occurring over one cell diameter. The apparent
difference in range would reflect the difference in timing
between these two effects and the intervening proliferation. This model is being tested by assessing the activity of a
membrane-tethered form of Hedgehog (Alexandre, 1999).
To sum up, in the bald area of abdominal segments, one cell
type forms in response to one signaling pathway while within
denticle belts, a rich pattern of cell types arise from juxtacrine
cell interactions initiated by the activation of distinct signaling
pathways. Some of these pathways are controlled by the
localized expression of segment polarity genes such as
wingless and hedgehog, while others are regulated by
downstream genes like veinlet and Serrate. Because
wingless and hedgehog are expressed first, they are effectively
at the top of the hierarchy and the knock-on effects of losing
hedgehog or wingless function explain the 'organizer activity'
of the parasegment boundary. Interestingly, the denticle
Hedgehog originating from the parasegment boundaries of
adjacent segments (and therefore, two parasegment boundaries)
are needed to provide the signals that pattern a single denticle
belt (Alexandre, 1999).
Dorsoventral axis formation in the Drosophila wing
depends on the activity of the selector gene apterous.
Although selector genes are usually thought of as binary
developmental switches, Apterous activity is found to be
negatively regulated during wing development by its target
gene dLMO. Apterous-dependent expression of Serrate and
fringe in dorsal cells leads to the restricted activation of
Notch along the dorsoventral compartment boundary. Evidence is presented that the ability of cells to participate in
this Apterous-dependent cell-interaction is under spatial
and temporal control. Apterous-dependent expression of
dLMO causes downregulation of Serrate and fringe and
allows expression of Delta in dorsal cells. This limits the
time window during which dorsoventral cell interactions
can lead to localized activation of Notch and induction of
the dorsoventral organizer. Overactivation of Apterous in
the absence of dLMO leads to overexpression of Serrate,
reduced expression of Delta and concomitant defects in
differentiation and cell survival in the wing primordium.
Thus, downregulation of Apterous activity is needed to
allow normal wing development (Milan, 2000).
Removing Apterous activity at different stages of wing
development shows that Ap is needed throughout larval stages
to confer dorsal cell identity, but its role in Notch activation
along the DV boundary is temporally and spatially modulated.
This can be explained in terms of changes in Serrate and fringe
expression. Some of the changes in Serrate and fringe
expression are caused by reducing Ap activity, whereas others
are Ap independent. In early second instar wing discs, Ap
activity is required in the entire dorsal compartment. Removing
Ap activity in mitotic recombination clones at this stage induces
Notch activation at the interface between wild-type and mutant
cells. This response is independent of the position of the clone
within the wing pouch. In early third instar wing discs, Ap-dependent
expression of Serrate and fringe is reduced by
dLMO. Serrate expression gradually becomes restricted to the
region near the DV boundary and, subsequently, by mid-third
instar is induced only in cells adjacent to the boundary. The effects of
removing Ap activity in clones reflects the gradual retraction of
Serrate expression toward the DV boundary. Clones of cells
lacking Ap activity induced in early third instar activate the
Notch pathway and induce Wg if they are located close to the
DV boundary. Clones located more proximally do not show this
response. This spatial difference can be
overcome by providing Serrate in proximal cells (Milan, 2000).
By mid-third instar, new Ap-independent patterns of Serrate
and fringe expression are observed. Serrate is expressed on
both sides of the DV boundary by the activity of Wg, and fringe
is expressed in four quadrants flanking the DV and AP
compartment boundaries. Maintenance of Notch activation
along the DV boundary is now under control of a feedback loop
between Wg and Serrate and Delta. Ap is no longer required for Notch
activation at the DV boundary and removing Ap activity no
longer leads to activation of the Notch pathway.
In the absence of dLMO, Ap activity remains at high early
levels as development proceeds. Serrate and fringe expression
remain high throughout the dorsal compartment and fail to
undergo normal modulation. In addition, Delta is not expressed
in dorsal cells. Ap-dependent repression of Delta at early
stages is needed to prevent ectopic activation of Notch in dorsal
cells, which are inherently Delta-sensitive due to the activity
of Fringe. Some of the defects observed in dLMO mutant wings are
correlated with excess Serrate activity and insufficient Delta
activity. In addition, abnormally high levels
of cell death in the dorsal compartment of the dLMO mutant
wing disc are due to excess Ap activity and this leads to
overall reduction in the size of the wing. These findings
indicate the need to downregulate Ap activity to allow normal
wing development. However, Ap activity continues to be
required for dorsal cell fate specification and for proper
adhesion of D and V wing surfaces. Thus it is proposed that
different target genes may be controlled at different levels of
Ap activity. Serrate, fringe and Delta may be regulated by a
higher level of Ap activity than the target genes involved in
surface apposition or fate specification. Temporal changes in
the level of Ap activity may be required to modulate activity
of different genes at different times to allow normal wing
development (Milan, 2000).
The Drosophila eye disc is a sac of single layer epithelium with two opposing sides: the peripodial membrane (PM) and the disc proper (DP). Retinal
morphogenesis is organized by Notch signaling at the dorsoventral (DV) boundary in the DP. Functions of the PM in coordinating growth and patterning of the
DP are unknown. The secreted proteins Hedgehog, Wingless, and Decapentaplegic are expressed in the PM. From there they control DP expression of the
Notch ligands Delta and Serrate. Peripodial clones expressing Hedgehog induce Serrate in the DP while loss of peripodial Hedgehog disrupts disc growth.
Furthermore, PM cells extend cellular processes to the DP. Therefore, peripodial signaling is critical for eye pattern formation and may be mediated by
peripodial processes (Cho, 2000).
Restricted localization of Hh-, Wg-, and Dpp-LacZ+ expressing cells along the DV axis in L1 discs suggests that these signals might act upstream of N. To test this idea, Hh activity was removed using a temperature-sensitive allele; Wg was ectopically expressed using hs-wg, or Dpp activity was removed by using a heteroallelic combination of the two dpp alleles, and then the expression patterns of the N ligands Dl and Ser were visualized in the eye discs. In L2 wild-type discs, Dl is preferentially expressed in the dorsal domain, while Ser is enriched along the DV midline of the DP. Both Dl and Ser are also present in the PM at a low level. In hhts2 mutants shifted to restrictive temperature during the early L1 stage, both Dl and Ser are uniformly expressed in dorsal and ventral domains. Ubiquitous Wg overexpression causes variable defects in Dl pattern such as significant reduction in the dorsal domain except near the margin or mislocalization to the ventral domain. Wg overexpression also causes mislocalization of Ser to the dorsal DP. dppe12/dppd14 mutant discs showed similar disruption of the DV-specific Dl and Ser pattern, indicating the necessity of Hh, Wg, and Dpp in DV patterning (Cho, 2000).
The complex interplay of Hh, Wg, and Dpp signaling has been studied for initiation and progression of the morphogenetic furrow. This study has examined much earlier stages of eye development to determine whether these same molecules organize DV patterning prior to retinal differentiation; it has been demonstrated that: (1) Hh, Wg, and Dpp display distinct DV expression patterns in the PM in early discs; (2) their signals are essential for domain-specific expression of Dl and Ser in the DP, and (3) signaling from the PM to DP is important for patterning in the DP. These findings provide a novel view of how eye discs are patterned, a model suggesting Hh, Wg, and Dpp signal from the PM to the DP by means of cellular processes (Cho, 2000).
The PM is an important source of inductive signals to control cell fates within the DP. According to the presented model, Hh acts differentially to localize Wg- and Dpp-expressing cells to the dorsal and ventral domains of the PM, respectively, in the L1 disc. Establishment of DV domains in the PM governs subsequent signaling from the PM to the DP for controlling the DV specificity and the level of Dl/Ser expression. This idea is supported by observations that ectopic Hh expression in the PM cells can induce Ser expression in the DP, consistent with spatiotemporal correlation of Hh and Ser expression pattern in the L1 and L2 discs (Cho, 2000).
Another important question raised by the prospect of interepithelial signaling is how the signals are transmitted from the PM cells to the DP. One possibility is that signaling molecules are secreted from PM cells directly to the underlying region of the DP. Alternatively, these molecules may be transported to the DP via peripodial trans-lumenal extensions that contact specific target cells or reach in the vicinity of target areas in the DP. Hh signaling may be mediated by peripodial processes, although the former possibility cannot be excluded. Ser expression in the DP induced by ectopic peripodial Hh signaling often extends beyond the region directly underneath the Hh+ PM cells. Hh may diffuse from the processes to reach other nearby DP cells. Alternatively, PM cells may extend longer processes than what can be detected in the fixed tissues. It is also possible that the inductive event occurs earlier when the two cell populations are in closer contact and subsequently become displaced relative to one another as the epithelium grows (Cho, 2000).
Recent studies have shown that disc cells send out long and thin cytoplasmic extensions, named cytonemes. Cytonemes are actin-based extensions that grow from the apical surface of the DP cells toward the signaling center, the anterior-posterior boundary of the wing disc. Some of the peripodial extensions described in this study also show cytoneme-like long and thin processes, although it is not known whether the processes are also actin-based. The peripodial processes observed can be readily seen in fixed tissues, unlike cytonemes that cannot be detected in fixed discs. Furthermore, cytonemes extend from the DP cells and grow on the apical surface of the DP, while peripodial processes extend from the apical surface of PM cells across the disc lumen to the DP. In addition, the observation of different shapes of processes suggests that peripodial processes exist in multiple types (Cho, 2000).
Inductive signaling between two cell layers is an important mechanism of morphogenesis in vertebrate development. For instance, BMP4 signaling between optic vesicle and surface ectoderm is important for lens induction in vertebrates. Wnt signaling between the ectoderm and the mesoderm is also crucial for proper dorsoventral limb patterning. First shown to occur during Drosophila leg disc regeneration and now in the eye, peripodial signaling to the DP may be analogous to such inductive signaling in vertebrates. This study illustrates a novel mechanism of interepithelial signaling between PM and DP layers and its importance in eye disc patterning. Significantly, ablation or genetic disruption of the PM also affects development of the DP, providing additional evidence for peripodial signaling. Precise localization of receptors and downstream components for Hh, Wg, and Dpp in early eye discs will help in understanding how these signals are transmitted between the PM and the DP (Cho, 2000).
The molecular basis of segmentation and regional growth during morphogenesis of Drosophila legs is poorly understood. four-jointed is not only required for these processes, but also can direct ectopic growth and joint initiation when its normal pattern of expression is disturbed. These effects are non-autonomous, consistent with the demonstration of both transmembrane and secreted forms of the protein in vivo. The similarities between four-jointed and Notch phenotypes led to an investigation of the relationships between these pathways. Surprisingly, it was found that although four-jointed expression is regulated downstream of Notch activation, four-jointed can induce expression of the Notch ligands, Serrate and Delta, and may thereby participate in a feedback loop with the Notch signaling pathway. four-jointed interacts with abelson, enabled and dachs, which suggests that one target of four-jointed signaling is the actin cytoskeleton. Thus, four-jointed may bridge the gap between the signals that direct morphogenesis and those that carry it out (Buckles, 2001).
Fj is regulated downstream of N signaling and many of the phenotypes observed with ectopic fj expression are similar to those seen upon ectopic activation of N. It is possible that these similarities might derive from a common molecular cause. For example, deregulation of N signaling may cause a deregulation of fj expression, which would then disrupt normal morphogenesis. Alternatively, since fj is known to have a transcriptional feedback on its own expression, perhaps it also participates in a feedback loop onto the N pathway such that misexpression of fj actually results in misexpression of activated N. The most likely target for such feedback would be the N ligands, since N is expressed widely in the disc but only becomes activated at the restricted positions of ligand expression (Buckles, 2001).
To investigate whether Fj feeds back onto the N signaling pathway, the expression of the N ligands Ser and Dl was examined in leg discs in which fj was ectopically expressed along the AP axis using ptc-Gal4-driven expression of UAS-fj. Such misexpression of fj results in severe truncation of the tarsus. ptc is expressed at highest levels along the AP boundary, with graded expression in the anterior compartment of the disc. Ectopic fj expression induces the expression of both Ser and Dl along the posterior edge of the fj-expressing stripe, and does so largely non-autonomously. The non-autonomy is consistent with biochemical data, and provides further evidence that Fj acts as a signaling molecule. Furthermore, the ectopic expression of Ser and Dl, leading to ectopic activation of N, could account for some of the observed effects of ectopic fj expression on leg development (Buckles, 2001).
The asymmetry of induction only along the border of highest expression raised the possibility that induction might only occur at sharp boundaries of expression, such as that on the posterior edge of the ptc domain. To test this, UAS-fj was expressed with two additional drivers, dpp-Gal4 and en-Gal4, which are both expressed at somewhat lower levels than ptc-Gal4. dpp-Gal4 is expressed within the anterior compartment of the leg disc, while en-Gal4 is expressed in the posterior compartment with a sharp boundary of expression along the AP border. Misexpression of fj under either driver produces truncations of the tarsus as well as apparent outgrowths and/or bifurcations of the distal leg. As with ptc-Gal4, both dpp- and en-Gal4-driven expression of fj induces expression of Ser in cells neighboring those expressing high levels of fj: at the posterior edge of the dpp domain and at the anterior edge of the en domain. Similar non-autonomous induction of Dl is observed with these drivers (Buckles, 2001).
Whether fj is required for normal Ser expression was investigated. Ser expression was examined in pupal leg discs homozygous mutant for fj. Expression of Ser is unaffected in all leg segments except for one: Ser expression is significantly reduced in the second tarsal segment. This finding is consistent with the observation that fj mutants have a partial or complete lack of the joint between the second and third tarsal segments and reduced growth within the fused segment (Buckles, 2001).
Since fj induces Ser expression non-autonomously, it was of interest to examine their endogenous expression patterns during development of the leg. Consistent with the inductive behavior observed, fj and Ser appear to be expressed in adjacent but largely non-overlapping stripes in tarsal segments 2-4 in the developing leg disc (Buckles, 2001).
Together, these results suggest that there is a feedback loop between N ligand expression and the N target gene fj. Fj appears to be necessary for the initiation, upregulation, or maintenance of Ser expression. Although fj is expressed in every tarsal segment, Ser expression is only affected by loss of fj in tarsal segment 2 (Buckles, 2001).
To begin to understand how Fj signaling affects such diverse processes as leg segmentation and growth, ommatidial rotation, and epithelial planar polarity, attempts were made to identify other genes with which Fj interacts. Some of the effects of Fj are likely to be due to its feedback onto the N signaling pathway, and this would presumably require a Fj signal transduction pathway. In addition, it is likely that Fj also functions independently of its regulation of Ser and Dl, since the loss-of-function and gain-of-function phenotypes of N ligands and Fj are not identical (Buckles, 2001).
Similar mutant phenotypes may indicate that the genes causing them may act in the same molecular pathway. dachs and abl mutant phenotypes imitate those of fj, thus both of these genes are attractive candidates for the fj signaling pathway. A major substrate for Abl kinase activity is the Ena gene product. Ena homozygotes are embryonic lethal and imaginal phenotypes are not known. However, Abl and Ena appear to function in the same pathway in Drosophila. Finally, given the molecular epistatic interactions observed between fj and Ser, whether fj and Ser interact genetically was tested (Buckles, 2001).
To test the relationships of these genes, two hypomorphic alleles of fj, fj4 and fjN7 were used. The majority of legs of fjN7 flies retain partial joints of a ball and socket morphology at the juncture between T2 and T3, while fj4 produces larger partial joints or complete joints at the T2/3 boundary. Introduction of one mutant copy of dachs, abl, ena, or Ser into these backgrounds significantly increases the severity of the hypomorphic phenotypes, while each of these genes by itself is wholly recessive in the leg. Thus, dachs, abl, ena, and Ser act as dominant enhancers of fj, suggesting these genes may be part of a common pathway or network (Buckles, 2001).
The morphogenetic gradient of Hh is tightly regulated for correct patterning in Drosophila and vertebrates. The Patched (Ptc) receptor is required for restricting Hh long-range activity in the imaginal discs. In this study, the different types of Hh accretion that can be observed in the Drosophila embryonic epithelial cells were investigated. In receiving cells, large apical punctate structures of Hh (Hh-LPSs) are not depending on the Ptc receptor-dependent internalization of Hh but rather reflect Hh gradient. By analyzing the dynamic of the Hh-LPS gradient formation, it was demonstrated that Hh distribution is strongly restricted during late embryonic stages compared to earlier stages. The up-regulation of Ptc is required for the temporal regulation of the Hh gradient. Dynamin-dependent internalization of Hh does not regulate Hh spreading but is involved in shaping Hh gradient. Hh gradient modulation is directly related to the dynamic expression of the ventral Hh target gene serrate (ser) and with the Hh-dependent dorsal cell fate determination. Finally, this study shows that, in vivo, the Hh/Ptc complex is internalized in the Rab7-enriched lysosomal compartment in a Ptc-dependent manner without the co-receptor Smoothened (Smo). It is proposed that controlled degradation is an active mechanism important for Hh gradient formation (Gallet, 2005).
To confirm that Ptc is involved in the temporal restriction of Hh movement, Hh distribution was analyzed in ptc mutant embryos and in embryos expressing ptc in en/hh cells. In ptc null embryos, the Hh gradient is impaired and Hh-LPSs distribution was found to be extended throughout the entire segment without restriction. In such embryos, ser expression is totally repressed in a manner similar to that seen under ubiquitous Hh expression. Note that the ectopic hh expressing source present in ptc mutant might also contribute to the broad distribution of Hh. When Ptc is expressed in en/hh expressing cells, the range of Hh-LPSs movement is limited to the vicinity of the producing cells. This effect is not due to a diminution of hh expression since it has been shown that Ptc does not affect hh transcription. Hence, Ptc might directly affect Hh-LPS range of action. Indeed, the slope of the Hh-LPSs gradient decreased sharply compared to wild-type stage 11 embryos. Interestingly, in these embryos, ser expression was extended correlating with the absence of Hh-LPS away from the source (Gallet, 2005).
Temporal regulation of Hh gradient is necessary because signaling requirements for Hh change in a time-dependent manner. One can suggest that, during early development, Hh acts at long range due to moderate levels of Ptc. Hh would easily overcome repression by the low concentrations of Ptc protein to prime a subset of ectodermal cells at a long distance to make them competent to respond to other signals. At later stages, Hh distribution is restricted and allows expression of ser and acts over a short range to induce rhomboid, both genes being necessary for ventral denticle specification (Gallet, 2005).
Segmentation is a developmental mechanism that subdivides a tissue into repeating functional units, which can then be further elaborated upon during development. In contrast to embryonic segmentation, Drosophila leg segmentation occurs in a tissue that is rapidly growing in size and thus segmentation must be coordinated with tissue growth. Segmentation of the Drosophila leg, as assayed by expression of the key regulators of segmentation, the Notch ligands and fringe, occurs progressively and this study defines the sequence in which the initial segmental subdivisions arise. The proximal-distal patterning genes homothorax and dachshund are positively required, while Distal-less is unexpectedly negatively required, to establish the segmental pattern of Notch ligand and fringe expression. Two Serrate enhancers that respond to regulation by dachshund are also identified. Together, these studies provide evidence that distinct combinations of the proximal-distal patterning genes independently regulate each segmental ring of Notch ligand and fringe expression and that this regulation occurs through distinct enhancers. These studies thus provide a molecular framework for understanding how segmentation during tissue growth is accomplished (Rauskolb, 2001).
In recent years the key role of the Notch signaling pathway in the segmentation and growth of the Drosophila leg has been established. Notch signaling must be localized within each leg segment to promote the formation of boundaries (joints) that separate each leg segment and to induce leg growth. This requirement for a segmentally repeated pattern of Notch activation is accomplished by restricting the expression of the regulators of Notch activation, Serrate, Delta and fringe, to one ring per segment. By examining the expression of the Notch ligands and fringe during leg development, it has been possible to determine the progressive order in which leg segmentation is established. At early third instar, a single ring of Serrate, Delta and fringe expression is present within the coxa. The next ring to arise is located within the presumptive femur. At mid third instar, expression arises within presumptive tarsal segments 2 and 5. Subsequent expression is observed in the tibia and more tarsal segments, such that ultimately, by the end of third instar, a ring of expression is present in each presumptive leg segment, adjacent to each prospective leg segment border. Thus, segmentation of the Drosophila leg occurs progressively and in a reproducible pattern (Rauskolb, 2001).
Previous studies investigating the expression of a reporter gene [E(spl)mß-CD2] regulated downstream of Notch activation led to the conclusion that the first segment boundary to form was between tarsal segments 4 and 5. Additional rings of expression were then observed in the tarsus and then eventually in all leg segments. This led to the suggestion that the first segmental boundaries to form correspond to the most distal segments. However, further examination of this reporter gene indicates that expression is observed in proximal cells prior to expression within the tarsus. Moreover, temperature shifts of a conditional Notch allele at different stages of development demonstrate that the temperature-sensitive period for Notch in proximal segmentation occurs before that in tarsal segmentation. The conclusion is reached that leg segmentation does not occur in a simple distal to proximal order, nor proximal to distal order, nor are the most proximal and distal segments established first and other segments added by intercalation. Rather, the establishment of Drosophila leg segmentation occurs in a complex sequence (Rauskolb, 2001).
A general theme in patterning during development is the subdivision of tissues initially by genes expressed in broad, partially overlapping domains, which through combinatorial control, subsequently regulate the expression of downstream genes to generate a repeating pattern. The studies presented here demonstrate that leg segmentation follows this same theme. The 'leg gap genes' Hth, Dac, and Distal-less are expressed in broad domains in the leg disc that encompass more than a single segment. Initially expression of these genes is largely nonoverlapping, but as the leg disc grows, the expression patterns of the leg gap genes change such that five different domains of gene expression are established. The analysis of the regulation of Notch ligand and fringe expression during leg development reveals two fundamental aspects of leg development. (1) These leg gap genes are key components in regulating the expression of the molecules controlling segmentation. Indeed, the effect of these leg gap genes on leg segmentation and growth can be accounted for by their regulation of Serrate, Delta and fringe expression. (2) The expression of each ring of Serrate, Delta and fringe is controlled by its own unique combination of regulators, apparently acting through independent enhancers (Rauskolb, 2001).
How do these three transcription factors regulate the formation of nine segments? Since the requirements for and the expression of the leg gap genes encompasses all leg segments, it is unlikely that there are additional leg gap genes yet to be identified. Rather, a collection of distinct combinatorial approaches is used to establish a segmental pattern of Serrate, Delta and fringe expression (Rauskolb, 2001).
In early third instar leg discs, there are two domains of gene expression: proximal cells express Hth and distal cells express Distal-less. Hth autonomously promotes the expression of Serrate, while Distal-less may prevent expression more distally, giving rise to a ring of expression in the coxa. Additionally, Distal-less-expressing cells may signal to the Hth-expressing cells to restrict Serrate expression to the distal edge of the Hth domain. As the leg disc grows, cells in an intermediate position, lying between the Hth and Distal-less domains, begin to express Dac. Dac, as shown in this study, is both necessary and sufficient to induce the expression of Serrate, Delta and fringe within the femur. Since they are not expressed in all Dac-expressing cells, other factors appear to be required to promote their expression in the proximal femur. The nonautonomous induction of Serrate expression by Hth suggests that this may be accomplished by a signal (X) emanating from the neighboring Hth-expressing cells. By mid third instar stages, expression of Serrate, Delta and fringe is also observed in tarsal segments 2 and 5, within cells expressing Distal-less but not Dac. Given that Distal-less is necessary and sufficient to repress their expression, Serrate, Delta and fringe expression within the tarsus appears to be induced by a mechanism that overrides the repressive effects of Distal-less. Subsequently, expression of Serrate, Delta and fringe is observed within the tibia, in cells expressing both Dac and Distal-less. Dac is necessary for expression of Serrate within the tibia, and its role here may be to overcome the repressive effects of Distal-less. It is also worth noting that the tibia ring of expression is not established at the time when cells first express both Dac and Distal-less. This may be because Dac levels may not be sufficiently high enough to overcome the repression by Distal-less. Clearly levels of Dac expression are critical because simply increasing Dac levels is sufficient to promote Serrate expression in cells already expressing endogenous levels of Dac. This observation can be explained if high levels of Dac expression in cells already expressing Dac override the function of inhibitory regulators of Serrate expression, such as Distal-less, where the expression of these genes overlap. Although late stages of leg segmentation were not investigated in this study, it has been noted that Hth, Dac and Distal-less are co-expressed in the presumptive trochanter late in leg development. It is thus hypothesized that Serrate, Delta and fringe expression is established by the combined activities of the three leg gap genes in the trochanter (Rauskolb, 2001).
Although these here have focused on the regulation of Serrate expression, it is thought that not only Serrate, but also Delta and fringe, receive primary regulatory input from the leg gap genes. Delta and fringe expression, like Serrate, is positively regulated by Dac. Moreover, Dl and fringe mutants have stronger leg segmentation phenotypes than Ser mutants, and thus Delta and fringe expression cannot simply be regulated downstream of Ser. The identification of two separate Ser enhancers, directing expression in the proximal versus distal leg, argues against Serrate being regulated downstream of Dl and fringe. Thus, the simplest model is that expression of all three genes is regulated directly by the leg gap genes. The regulation of Serrate, Delta and fringe expression in each segment appears to occur through independent and separable enhancer elements, supported by the analysis of the Ser reporter genes. This is reminiscent of what occurs during Drosophila embryonic segmentation, where separable enhancer elements direct different stripes of pair-rule gene expression (Rauskolb, 2001).
Importantly, Notch signaling may actually coordinate progressive segmentation of the leg with leg growth. For example, in early leg discs there is a single ring of Serrate expression within the coxa, in Hth-expressing cells immediately adjacent to Dac-expressing cells. However, by the time the femur ring arises, the coxa ring of Serrate expression has been displaced and is no longer within cells immediately adjacent to the Dac-expressing cells; rather, there are Hth-expressing cells lying in between that do not express Serrate. Thus, it is postulated that once Serrate, Delta and fringe expression is established within the coxa, Notch is activated, which promotes local cell proliferation, thereby displacing the coxa ring. This then allows for the femur ring of expression to be established in cells that are not immediately adjacent to the coxa expression ring. This mechanism also requires that once a ring of ligand expression is established in a particular segment, this expression must be maintained such that it is not influenced by later alterations in relation to leg gap gene expression. This maintenance could be accomplished by a feedback loop between Notch activation and ligand expression, similar to what has been observed during late wing development, where Notch activation cell autonomously represses ligand expression and nonautonomously induces ligand expression in flanking cells by regulating the expression of a signaling molecule. Preliminary studies have indicated that Notch activation can influence Notch ligand expression in the developing leg (Rauskolb, 2001).
Most of the tarsus of the Drosophila leg derives from cells expressing Distal-less, but not Dac or Hth. Surprisingly, the studies presented here have shown that Distal-less actually represses Notch ligand expression. This negative regulatory role for Distal-less contrasts with the positive promoting role of Dac and Hth, and further indicates that a distinct molecular mechanism must promote segmentation within the tarsus. One key gene is spineless-aristapedia (ss), since simple, unsegmented tarsi develop in ss mutant flies. Moreover, ss regulates the expression of bric-à-brac (bab), which is also required for the subdivision of the tarsus into individual segments. Together, ss and bab must, in some way, ultimately overcome the repression of Notch ligand and fringe expression by Distal-less. If the sole function of ss and bab is to overcome the inhibitory effects of Distal-less, then in the absence of ss and/or bab, Serrate expression is expected to remain repressed (Rauskolb, 2001).
Intriguingly, the only notable variation between insect species is in the number of tarsal segments, with an unsegmented tarsus believed to be the ancestral state. Thus, the combinatorial regulation of segmentation by the leg gap genes may represent an ancient mechanism common to all insect species, a hypothesis supported by the conserved expression of Hth, Dac and Distal-less in the developing legs of many insect species (Rauskolb, 2001 and references therein).
Notch (N) activation at the dorsoventral (DV) boundary of the Drosophila eye is required for early eye primordium growth. Despite the apparent DV mirror symmetry, some mutations cause a preferential loss of the ventral domain, suggesting that the growth of individual domains is asymmetrically regulated. The Lobe (L) gene is required non-autonomously for ventral growth but not dorsal growth; it mediates the proliferative effect of midline N signaling in a ventral-specific manner. L encodes a novel protein with a conserved domain. Loss of L suppresses the overproliferation phenotype of constitutive N activation in the ventral, but not in the dorsal eye, and gain of L rescues ventral tissue loss in N mutant background. Furthermore, L is necessary and sufficient for the ventral expression of a N ligand, Serrate (Ser), which affects ventral growth. These data suggest that the control of ventral Ser expression by L represents a molecular mechanism that governs asymmetrical eye growth (Chern, 2002).
The L gene was first reported in 1925 by Morgan and has been commonly used as a second chromosome dominant marker. However, mechanisms that underlie its growth defect are little understood. With variable severities and penetrances, all L alleles examined (L1, L2, L4, L5 and Lsi) as heterozygotes exhibit a nick near the anterior midline of the eye, and the overall size of the eye is slightly reduced. As homozygotes, eye size is greatly reduced, primarily in the ventral domain. Lsi allele typifies the observed phenotypes with the highest penetrance. Homozygous Lsi animals show a preferential loss of the ventral eye with complete penetrance, and ~70% of Lsi/+ animals have an anterior nick in one or both eyes. The preferential loss of the ventral eye is also apparent in the eye imaginal disc morphology. Importantly, since homozygous Lsi mutants are viable and its half-eye mutant phenotype is indistinguishable from that of Lsi over deficiency chromosomes, this suggests that Lsi is a strong eye-specific allele that minimally affects the development of other tissues (Chern, 2002).
To assess the extent of ventral eye loss in Lsi homozygotes, an enhancer trap line, mirrB1-12 (mirr-lacZ), which has a dorsal-specific expression of white (w) reporter gene, was used. In w-; Lsi/Lsi; mirrB1-12/+ flies, the overall eye size is reduced, and all but one or two rows of remaining ommatidia are w+, suggesting that most, if not all, of the ommatidia are dorsal. The dorsal polarity was confirmed in adult-eye sections. Eye imaginal discs from staged w-; Lsi/Lsi; mirrB1-12/+ larvae showed ventral domain reduction starting at early second instar, indicating that L functions are required for early eye development (Chern, 2002).
Clones of L- cells were generated by mitotic recombination, using a loss-of-function allele, Lrev6-3. Homozygous Lrev6-3 embryos with no detectable L protein expression fail to complete germ-band retraction and show no cuticle formation. Lrev6-3 clones cause distinct eye phenotypes depending on the time of clone induction and the location of the clones. In one scheme, clones were induced at the first instar stage. In the resulting third instar eye discs that contained ventral Lrev6-3 clones, the ventral eye disc is greatly reduced, and to a large extent the Lrev6-3/+ and +/+ tissue disappear together with the Lrev6-3/Lrev6-3 tissue. By contrast, dorsal clones of considerable size do not cause obvious size reduction in the dorsal eye, nor do the mutant clones significantly affect the ensuing photoreceptor differentiation and polarity determination. Consistent with the eye disc phenotype, adult mosaic eyes have a relatively normal appearing dorsal domain, while most of the ventral region is replaced by the cuticle. In related experiments, mitotic recombination induced at late second and third instar stages generated multiple clones in both dorsal and ventral domains but did not result in any obvious eye defects, suggesting an early, transient requirement of L. All together, studies of Lsi phenotype and Lrev6-3 clonal phenotype indicate that L is non-autonomously required for the ventral eye growth but not so in the dorsal, and its functions are required during early stages of eye development (Chern, 2002).
It is known that N activation at the DV boundary is vital for eye disc growth. Since L is required specifically for ventral growth, it raises the possibility that L may mediate the proliferative effect of midline N signaling in the ventral eye. The Gal4-UAS system was used to test this hypothesis. Overexpression of a constitutively active N (Nintra) by the dpp-Gal4 driver, which drives expression along the posterior edge of the eye disc, causes gross overgrowth of the eye in both dorsal and ventral domains. Reducing L gene dose strongly suppresses the ventral overgrowth but has much less of an effect on dorsal overgrowth. This ventral-specific suppression of N gain-of-function phenotype suggests that L acts downstream of N (Chern, 2002).
In contrast to N-induced overgrowth, eliminating N signaling by expressing a dominant-negative form of N (NDN) using the eyeless (ey)-Gal4 driver consistently results in small-eye or no-eye. ey-Gal4 drives Gal4 expression in early eye discs and anterior to the furrow in the third instar discs. Co-expression of L and NDN partially suppresses this NDN overexpression phenotype in the ventral domain: ventral eye was selectively restored in close to 20% of ey-Gal4/UAS-NDN UAS-L animals. The size of the restored ventral eye was either smaller or equal to the reduced dorsal eye, and in no instances was ventral tissue detected without the presence of at least some dorsal tissue. The presence of residual dorsal eye indicates that NDN overexpression may not completely eliminate endogenous N functions. It also suggests that N activity, even at a low level, is a prerequisite for L to induce ventral proliferation (Chern, 2002).
Given that the requirement of L functions is early and transient, the suppression by L of NDN phenotype may be specific to undifferentiated cells. This is indeed the case. NDN was overexpressed using GMR-Gal4 that induces Gal4 expression in all cells posterior to the furrow. GMR-Gal4/UAS-NDN animals show a rough eye phenotype with a relatively normal eye size, and the eye roughness is not suppressed by overexpression of L in GMR-Gal4/UAS-NDN UAS-L animals (Chern, 2002).
If L and N act in the same pathway, transheterozygous mutations of these two genes may result in enhanced phenotypes. Lsi/+ flies have nicks at the anterior edge of the eye, but the defect is not so severe to result in half-eyes. Loss of one copy of N does not cause visible eye defects. Transheterozygote N264-47/+; Lsi/+ adults, however, had half-eyes in one or both eyes with approximately 50% penetrance. Similar enhancement is observed of L phenotype by mutations in Enhancer of split, a major downstream effector of N signaling. In summary, genetic interactions between L and N support the hypothesis that L mediates the proliferative effect of N signaling specifically in the ventral domain (Chern, 2002).
It is likely that L interacts with other ventral-specific genes, and one candidate gene is Ser, a N ligand, whose expression is ventrally enriched in the wild-type first instar eye disc. Ser is required for eye development; Ser loss-of-function mutants have small eyes. To understand possible regulatory relations between L and Ser, Ser expression was examined in first instar eye discs from Lsi homozygotes. In these eye discs, ventral Ser expression is greatly reduced, but notably the expression along the DV boundary is not affected. A Ser-lacZ enhancer trap line and an anti-Ser antibody were both used to detect Ser expression in these Lsi homozygotes eye discs, and similar Ser expression patterns were observed. This observation suggests that L might promote ventral Ser expression except in regions near the DV boundary. In addition, since the Lsi homozygote eventually loses its ventral eye, loss of ventral Ser expression in these mutants suggests that Ser may positively regulate ventral eye growth (Chern, 2002).
L null clones were induced to further test the hypothesis that L is a region-specific, positive regulator of Ser expression in first instar eye discs. Lrev6-3 clones alter Ser expression in a position-dependent way. Ventral L- clones away from the DV boundary show decreased Ser expression within the clone, but clones near the posterior DV boundary have no such effects. The effect of L- cells on Ser expression appear to be restricted within the clone since Ser expression outside of the clone is not visibly affected. To test if L positively regulates Ser expression, the flp-out system was used to generate clones of cells that overexpress L. In dorsal and ventral domains of first instar eye discs, L flp-out clones either induce or upregulate Ser expression, respectively. In summary, L is crucial in maintaining ventral Ser expression levels (Chern, 2002).
To understand the role of Ser in eye growth, Ser-null (Serrev2-11) mutant clones were examined in eye discs and adult eyes. No obvious defects were found in the size of the eye disc or photoreceptor differentiation. This lack of Ser-null clone phenotype implies that either the Ser protein is diffusible, or Ser is functionally redundant. In order to remove more of the wild-type Ser functions, flp-out clones expressing a diffusible, truncated form of Ser (SerDN) were generated. SerDN consists of Ser extracellular domain but lacks the transmembrane domain and is capable of antagonizing wild-type Ser functions (Chern, 2002).
Eye discs that contain SerDN flp-out clones are variably reduced. Three kinds of phenotypes are observed and might be attributed to slight variations in the timing of clone inductions and the location of the clones:
Taken together with the regulatory relations between L and Ser, these results suggest that loss of ventral Ser expression probably contributes to L ventral eye loss (Chern, 2002).
The putative L protein is 562 amino acids long and contains a poly-glutamine rich region and a C terminus that shares significant sequence similarities with novel insect, mouse and human proteins of unknown functions. A polyclonal antibody detected a single band of ~60 kDa on a Western blot prepared from third instar eye imaginal discs. Furthermore, this antibody revealed no detectable level of L protein expression in null animals. To examine L expression pattern, L transcript and protein were detected by mRNA in situ hybridization and by the polyclonal antibody. L is ubiquitously expressed in first instar eye discs. In the third instar disc, the RNA transcripts are detected in the antenna disc and in undifferentiated cells anterior to the furrow in the eye disc. By comparison, the protein can be readily detected in the antenna and anterior to the furrow, but a much lower level of expression is also present posterior to the furrow (Chern, 2002).
The putative L protein contains a poly-glutamine rich region. The C terminus covering 67 amino acids is conserved in homologs from various species; bee (BI509118) (43% identical; 63% positive), mouse (AK003638) and human (BC007416) (37% identical; 53% positive). In the last 30 amino acids, 56% are identical. The conserved L sequence suggests that its mammalian counterparts may play similar functions in mediating N signaling, although the precise function of the conserved domain is not known. The striking domain specificity of L-mediated growth may be the result of various mechanisms. It is possible that signaling molecules present in the ventral domain are different from the ones present in the dorsal domain. Signaling molecules may be selectively active in one domain but not the other, as in the case of dpp signaling in the wing disc. L may cooperate with other ventral-specific genes to transduce the N signaling, or the expression of L may be transiently ventral specific in the early eye disc. More than one mechanism may contribute to the domain specificity of L functions (Chern, 2002).
Previous experiments have shown that growth of neighboring, symmetrical domains may be independently controlled. In Drosophila wing discs, increased expression of hedgehog along the anteroposterior boundary causes anterior wing overgrowth but has no effects on the posterior wing; ubiquitous overexpression of Ser increases the ventral wing tissue but not the dorsal. Additionally, there are other Drosophila eye mutants that show preferential reductions of the ventral eye, such as wg mutants and dpp mutants. However, dominant mutation Rough eye suppresses these 'furrow stop' mutant phenotypes of dpp and wg but not the L phenotype, suggesting fundamental differences in nature and function between L and furrow stop mutants (Chern, 2002).
Clonal study shows that L null clones have striking domineering non-autonomous effects, such that the viability of wild-type tissue immediately adjacent to L mutant clones are severely reduced. Nevertheless, this non-autonomous deleterious effect is limited to the ventral domain, since clones abutting the DV boundary do not seem to affect dorsal cell viability. This domineering non-autonomous effect may be the result of interspersed L null clones disrupting the physical integrity of the imaginal disc epithelium, causing the disc to fall apart. It is also possible that L is redundant in the dorsal domain, thus loss of L can be compensated by another dorsal-specific gene. Another possibility that is favored incites a failure of the clone cells to send out a locally acting growth signal. Since the data indicate that L is a regulator of Ser expression, could Ser be this local-acting, diffusible factor?
Homozygous Ser mutants have small eyes, indicating that Ser is required for proper eye growth. However, removing Ser in clones of cells does not result in mutant eye phenotypes. These observations are consistent with Ser being a diffusible factor, but other possibilities exist (Chern, 2002).
First, there may be other functionally redundant Ser-like molecules, but no candidates have yet been identified. Second, in Ser-expressing cells, Ser may autonomously induce the expression of diffusible signaling molecules that act non-autonomously. Ligand and receptor interactions within the membrane of the same cell have been demonstrated in the case of N and Dl. N and Dl can physically associate within the membrane of a single cell, and the expression level of Dl in a cell can modulate its own N response. In this manner, Ser-N interaction may lead to the expression of diffusible factors that rescue clones of Ser- cells. Third, the ability of adjacent wild-type cells to rescue Ser- cells suggests that Ser protein may be diffusible. This is consistent with low levels of Ser expression observed in L- clones; and in anti-Ser antibody staining, intense, dotty cytoplasmic staining, possibly secretory vesicles were consistently observed. However, the presence of secreted Ser has yet to be confirmed, even though diffusible Dl has been detected in Drosophila extract (Chern, 2002).
The domain-specificity of L phenotype indicates that the eye disc is partitioned, and the growth of individual domain is differentially regulated. Loss of the ventral eye in L mutants does not seem to affect DV boundary formation or the associated midline N activation, because disruptions of either of these events would result in abnormal dorsal growth. Additional data suggest that L does not affect the initial DV domain specification: (1) L is functionally downstream of N activation; (2) L mutation does not affect Ser expression at the DV boundary; and (3) domain-specific expression patterns of dpp, fng and wg are not affected in the first instar L mutant eye discs (Chern, 2002).
Consistent with this model, it is proposed that in the seemingly homogenous Ser-expressing, first instar ventral domain, there are actually two distinct groups of Ser-expressing cells: ventral midline cells abutting the dorsal midline cells, and the rest of the ventral cells. Their putative functions are different and their Ser expression is independently regulated. In the ventral midline cells, Ser is involved in setting up the DV boundary, and its expression is regulated by the Ser-N-Dl positive-feedback loop. The midline Ser expression can be further modified by Fng and Hedgehog, both of which can induce Ser expression only near the DV boundary but not elsewhere in the eye field, emphasizing again the distinctiveness of these midline cells (Chern, 2002).
By comparison, in the rest of the ventral domain, Ser is directly involved in controlling local growth. Loss of Ser in the ventral domain causes ventral-specific growth defects similar to the loss of L. Ser expression in the ventral domain may not be sustained by the Ser-N-Dl loop, since ventral Fng inhibits potential Ser-N interaction which is necessary to initiate the positive feedback loop. Instead, ventral Ser expression is regulated by L (Chern, 2002).
The data suggest the eye primordium is partitioned into dorsal, midline and ventral domains with different gene expression and growth properties. It highlights the importance of local cellular context in interpreting signals released from the domain boundaries and shows that the growth of symmetrical domains may be asymmetrically regulated. The model may also be applicable to the development of other imaginal discs as well as other developmental systems (Chern, 2002).
The segmentation of the proximal-distal axis of the Drosophila leg depends on the localized activation of the Notch receptor. The expression of the Notch ligand genes Serrate and Delta in concentric, segmental rings results in the localized activation of Notch, which induces joint formation and is required for the growth of leg segments. This study reports that the expression of Serrate and Delta in the leg is regulated by the transcription factor genes dAP-2 and defective proventriculus. Previous studies have shown that Notch activation induces dAP-2 in cells distal and adjacent to the Serrate/Delta domain of expression. Serrate and Delta are ectopically expressed in dAP-2 mutant legs, and Serrate and Delta are repressed by ectopic expression of dAP-2. Furthermore, Serrate is induced cell-autonomously in dAP-2 mutant clones in many regions of the leg. It was also found that the expression of a defective proventriculus reporter overlaps with dAP-2 expression and is complementary to Serrate expression in the tarsal segments. Ectopic expression of defective proventriculus is sufficient to block joint formation and Serrate and Delta expression. Loss of defective proventriculus results in localized, ectopic Serrate expression and the formation of ectopic joints with reversed polarity. Thus, in tarsal segments, dAP-2 and defective proventriculus are necessary for the correct proximal and distal boundaries of Serrate expression and repression of Serrate by defective proventriculus contributes to tarsal segment asymmetry. The repression of the Notch ligand genes Serrate and Delta by the Notch target gene dAP-2 may be a pattern-refining mechanism similar to those acting in embryonic segmentation and compartment boundary formation (Ciechanska, 2007).
Serrate is expressed throughout the dorsal compartment of wing discs (Diaz-Benjumea, 1995 and Kim, 1995). Serrate activity is required to induce wingless expression in the ventral compartment through Notch receptor (Diaz-Benjumea, 1995).
The role of the Notch and Wingless signaling pathways has been investigated in the maintenance of wing margin identity through the
study of cut, a homeobox-containing transcription factor and a late-arising margin-specific marker. By late third instar, a tripartite
domain of gene expression can be identified in the area of the dorsoventral compartment boundary, which marks the presumptive wing
margin. A central domain of cut- and wingless-expressing cells is flanked on the dorsal and ventral side by domains of cells
expressing elevated levels of the Notch ligands Delta and Serrate. Cut acts to maintain margin wingless expression,
providing a potential explanation for the cut mutant phenotype. Notch, but not Wingless signaling, is autonomously required for cut expression. Rather, Wingless is required indirectly for cut
expression; the results suggest this requirement is due to the regulation by wingless of Delta and Serrate expression in cells flanking
the cut and wingless expression domains. Delta and Serrate play a dual role in the regulation of cut and wingless
expression. Normal, high levels of Delta and Serrate can trigger cut and wingless expression in adjacent cells lacking Delta and
Serrate. However, high levels of Delta and Serrate also act in a dominant negative fashion, since cells expressing such levels cannot
themselves express cut or wingless. It is proposed that the boundary of Notch ligand along the normal margin plays a similar role as part
of a dynamic feedback loop that maintains the tripartite pattern of margin gene expression (Micchelli, 1997).
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Thus the combined effects of Notch and its target genes cut and wingless regulate the expression
of Notch ligands, which restricts Notch activity to the dorsoventral boundary (de Celis, 1997).
Serrate:
Biological Overview
| Evolutionary Homologs
| Protein Interactions
| Developmental Biology
| Effects of Mutation
| References
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