Serrate
Asymmetric divisions allow a precursor to produce the four distinct cells of Drosophila sensory organ lineages (SOLs). The sensory organ precursor (SOP) first divides into two different secondary precursor cells, IIA and IIB, which gives rise to one shaft-producing cell (trichogen) and one socket-producing cell (tormogen), and one neuron and one sheath cell (thecogen), respectively. Although this process requires cell-cell communication via the Notch (N) receptor, mitotic recombination that removes the N ligand Delta (Dl) or Serrate (Ser) in the SOL has mild or no effect. N mutant clones generated on the central region of the adult scutum are devoid of any external bristle structures, such as shafts and sockets, similar to the Nts mutant phenotype at a restrictive temperature. Whereas loss of N function during the process of lateral inhibition produces supernumerary SOPs, this balding phenotype is probably due to the requirement of N in asymmetric divisions. Without N activity the supernumerary SOPs divide symmetrically, giving rise to two IIB cells and, consequently, no external sensory structures. Dl clones typically produce a tuft of densely packed bristles in the interior of the clone. These tufts of bristles are likely due to a failure of lateral inhibition, resulting in overproduction of SOPs. The presence of the external bristle structures in these Dl mutant clones indicates that, unlike N clones, most of the supernumerary SOPs in the Dl mutant clones produce IIA cells that divide to form shaft and socket cells. Clones homozygous for three Ser null alleles produce normal external bristle structures. In contrast, clones with loss of both Dl and Ser function produce epidermal cells but not external bristle structures. This balding phenotype is clearly different from the phenotypes of the Dl or Ser mutant clones but is indistinguishable from that of N mutant clones, suggesting that Ser and Dl have overlapping functions in the N signaling pathway. Dl and Ser also have redundant functions in patterning wing veins. In contrast, Dl and Ser are known to serve distinct functions in specifying dorsal-ventral compartment boundary of the wing (wing margin). Ser in dorsal cells signals to N in ventral cells, and Dl in ventral cells signals to N in dorsal cells. For Dl and Ser to provide distinct signals from one compartment to the other without generating signals among cells within the same compartment, it may be necessary to involve other factors such as those encoded by the dorsally expressed gene fringe (fng), which inhibits a cell's ability to respond to Ser and potentiates a cell's response to Dl. It is concluded that Dl and Ser are redundant in mediating signaling between daughter cells to specify their distinct sensory organ cell fates (Zeng, 1998).
Ser mutant larva often die, exhibiting a failure to differentiate the anterior spiracles, poorly developed mouth-hooks and a severe reduction in the size of wing and haltere primordia. The few mutants that successfully pupate develop into pharate adults, almost completely lacking in wings and halteres (Speicher, 1994). The dominant Ser mutation causes a gap in the posterior wing tip and margin, and a portion of the blade (Jack, 1992).
Flies hetero- or homozygous for the dominant
mutation SerD exhibit scalloping of the wing margin due to cell death during pupal stages. SerD is
associated with an insertion of the transposable element Tirant in the 3' untranslated region of the
gene, resulting in the truncation of the Ser RNA, thereby eliminating putative RNA degradation
signals located further downstream. This leads to increased stability of Ser RNA and higher levels
of Serrate protein. Wing discs of SerD third instar larvae exhibit additional Serrate protein
expression in the edge zone of the future wing margin, where it is normally not detectable. Expression of wing margin specific genes, such as cut and wingless, is repressed in these
cells (Thomas, 1995).
Ectopic expression of Serrate during wing development induces ectopic outgrowth of ventral wing tissue and formation of an additional wing margin. In order for Serrate to elicit these responses the concomitant expression of wingless seems to be required. Ectopic expression of Delta provokes wing outgrowth and induction of a new margin, both on the dorsal and ventral side. Serrate acts downstream of apterous and induces expression of wing margin patterning genes. Serrate also has the potential to repress margin-specific genes such as wg and cut. This repression results in the failure to differentiate a proper wing margin, visible as a notch in the distal-most part of the wing margin. Thus ectopic Serrate provokes one of two responses, depending on the time and place of expression: outgrowth of wing tissue and induction of a new margin, or repression of margin specific genes that results in a nick in the wing margin. Actions of both Serrate and Delta are mediated by Notch, suggesting that the effects of the two Notch ligands depend on the cellular context, since the capability to activate Notch is spatially and temporally restricted, and expression of ligands at other times and in other places results in repression of Notch activity (Jönsson, 1996).
To ask whether the wing abnormalities caused by reducing Beadex levels
might be due to an effect on Apterous activity, the effects of
the Bxhdp excision mutants were examined on Ap target gene
expression. In early third-instar fng-lacZ and Serrate (Ser)
are expressed evenly throughout the dorsal compartment of the wing disc
and are thought to be regulated by Ap. In Bxhdp mutant
discs, the size of the dorsal compartment is considerably reduced,
consistent with the small wing phenotype. fng-lacZ expression is not affected in
Bxhdp discs. Ser expression is elevated
in the dorsal compartment and does not resolve normally into stripes
along the DV boundary and wing veins. Ser expression in the
ventral compartment appears normal. The stripes of Ser expression along
the DV boundary and wing veins are both dorsal and ventral and are under different regulation than the early
dorsal-specific domain. The abnormal pattern of Ser in the dorsal compartment of the
Bxhdp may be due to superimposition of the early and
late expression patterns. It is suggested that this reflects a failure to
down-regulate Ap activity as the disc matures. To ask whether elevated Ser levels might contribute to the defects
observed in Bxhdp mutant wings, Ser was overexpressed
in the dorsal compartment of an otherwise wild-type disc using
ap-gal4 to direct UAS-Ser expression. The
resulting wings are small and show thickened veins but do not show the
abnormalities in vein pattern observed in the Bxhdp
mutant wings. Overexpression of fng using ap-gal4 in
a wild-type background produces no phenotype. These
observations suggest that Ser overexpression contributes to the
abnormalities observed in Bxhdp mutant wings but
that there are likely to be additional factors. Thus, both gain-of-function and loss-of-function Bx mutant phenotypes can be attributed to abnormal regulation of Ap activity. It is
concluded that Ap induces dLMO expression in the wing disc and that dLMO then
functions as part of a feedback system to regulate the level of Ap activity (Milan, 1998).
Serrate is an essential gene in Drosophila melanogaster, best known for the Ser dominant (SerD)
allele and its effects on wing development. Animals heterozygous or homozygous for SerD are viable
and exhibit loss of wing margin tissue and associated bristles and hairs. The Beaded of Goldschmidt
(BdG) allele of Ser, when heterozygous to wild type, will also produce animals exhibiting loss of wing
margin material. However, animals homozygous for BdG exhibit a larval lethal phenotype comparable
to animals homozygous for loss-of-function Ser alleles. BdG is a partial duplication of the Ser locus
with a single 5' Ser-homologous region and two distinct 3' regions. Meiotic recombination between BdG
and a wild-type Ser chromosome demonstrates that only one DNA lesion, caused by the insertion of a
transposable element into the coding regions of the Ser transcript, appears capable of generating
BdG phenotypes. Due to the insertion, the protein product is predicted to be prematurely truncated
and lack an extracellular cysteine-rich region along with the transmembrane and intracellular domains
found within the normal SERRATE (SER) protein. The loss of these protein domains apparently
contributes to the antimorphic nature of this mutation (Hukriede, 1997a).
Specification of the dorsal-ventral compartment boundary in the developing Drosophila wing disc requires activation of Notch from
its dorsal ligand Serrate and its ventral ligand Delta. Both Notch ligands are required in this process: one cannot be
substituted for the other. In the wing disc, expression of BD(G), a dominant-negative, truncated form of Serrate, is capable
of inhibiting Notch activation in the ventral but not the dorsal compartments. BD(G) can act as a general
antagonist of both Serrate and Delta mediated Notch interactions. However, BD(G) retains the Serrate protein domain
targeted by Fringe, hence BD(G)'s antagonistic effects are restricted in the dorsal wing disc. Implicit in these results is the suggestion that binding of a ligand to Notch is not sufficient for Notch activation. The specificity of the Notch signal generated by interactions with Serrate and Delta originates from regions residing outside of the Notch binding domains of these molecules; other properties attributable to Notch ligands are required for Notch activation. Thus, ligand
binding to Notch is a necessary but insufficient step toward Notch activation (Hukriede, 1997b).
The functions of artificially constructed secreted forms of the two known Drosophila Notch ligands, Delta and Serrate, were examined by expressing
them under various promoters in the Drosophila developing eye and wing. The phenotypes associated with the expression of secreted
Delta (DlS) or secreted Serrate (SerS) forms mimic loss-of-function mutations in the Notch pathway. Both genetic interactions
between DlS or SerS transgenics and duplications or loss-of-function mutations of Delta or Serrate indicate that DlS and SerS behave
as dominant negative mutations. Expression of DlS and SerS in the eye results in a rough eye phenotype. This phenotype is enhanced by loss-of-function Delta and gain-of function Suppressor of hairless. These observations were extended to the molecular level by demonstrating that the expression of
Enhancer of split mdelta, a target of Notch signaling, is down-regulated by SerS. The antagonistic nature of the two mutant secreted
ligand forms in the eye is consistent with their behavior in the wing, where they are capable of down-regulating wing margin specific
genes in an opposite manner to the effects of the endogenous ligands. For example, wingless expression is down-regulated where a SerS expressing stripe crosses the dorsal/ventral boundary. The secreted ligands also interfere with wing vein specification. This analysis uncovers secreted molecular antagonists of Notch signaling and
provides evidence of qualitative differences in the actions of the two ligands DlS and SerS (Sun, 1997).
Dfrizzled3 is a novel member of the Frizzled family of seven-pass transmembrane receptors. Like Dfz2, Dfz3 is a target gene of Wingless (Wg) signaling, but in contrast to Dfz2, it is activated rather than repressed by Wg signaling in imaginal discs. Dfz3 is not required for viability but is necessary for optimal Wg signaling at the wing margin. Dfz3 was identified by characterizing a P-element line from a large scale Gal4 enhancer trap screen that allows
direct visualization of gene expression patterns in living
flies. A Gal4 insert found in the cytological position 1C exhibits an adult expression pattern resembling that of wg. The Gal4
expression pattern of this line has been visualized by a UAS-lacZ
reporter gene. Depending on the tissue analysed, Dfz3-Gal4 is expressed in a broad domain centered over, or in a domain coinciding with, the wg expression domain. Dfz3 is expressed throughout the wing pouch but appears to be upregulated by Wg signaling at the presumptive wing margin and
in a ring around the pouch. In the notum the expression is
similar to the thoracic expression of wg. In the leg disc Dfz3 is
expressed in a broad ventral wedge centered on the wg
domain. Dfz3 expression in the eye disc is also coincident
with wg expression and can be detected at the dorsal and
ventral margins, which give rise to the head capsule Evidence supporting a functional role for Dfz3 in Wg
signaling comes from a genetic interaction with Serrate
(Ser), a gene that encodes a Notch ligand involved in
establishing wg expression in the wing margin. SerD
is a dominant
mutation that results in reduced wg expression in wing
margin cells. Dfz3 mutations enhance
the SerD
phenotype in a dose-dependent manner.
With one copy of Dfz3 removed the SerD
wings show stronger notching and a loss of wing margin bristles. Removing
the second copy of Dfz3 enhances this phenotype further and
results in additional posterior margin notching. It is suggested that Dfz3 may
function in concert with Dfz2 and Fz to transduce or transport the Wg signal in imaginal discs (Sivasankaran, 2000).
Drosophila larval hemocytes originate from hematopoietic organs called lymph glands. These are composed of paired lobes located along the dorsal vessel. Two mature blood cell populations are found in the circulating hemolymph: the macrophage-like plasmatocytes, and the crystal cells that contain enzymes of the immune-related melanization process. A third class of cells, called lamellocytes, are normally absent in larvae but differentiate after infection by parasites too large to be phagocytosed. Evidence is presented that the Notch signaling pathway plays an instructive role in the differentiation of crystal cells. Loss-of-function mutations in Notch result in severely decreased crystal cell numbers, whereas overexpression of Notch provokes the differentiation of high numbers of these cells. In this process, Serrate, not Delta, is the Notch ligand. In addition, Notch function is necessary for lamellocyte proliferation upon parasitization, although Notch overexpression does not result in lamellocyte production. Finally, Notch does not appear to play a role in the differentiation of the plasmatocyte lineage. This study underlines the existence of parallels in the genetic control of hematopoiesis in Drosophila and in mammals (Duvic, 2002).
Crystal cells differentiate within the larval hematopoietic organ, the lymph glands, and the effect of the various mutations on differentiation in situ were examined. For this, an antibody raised against dipteran prophenoloxidase (proPO), a zymogen required for melanization reactions, was examined. In Drosophila, proPO is produced and stored in crystal cells and is released during host defense reactions. It was first noted that, in wild-type larvae, proPO is synthesized during the differentiation of crystal cells within the hematopoietic organ. Interestingly, a gradient of differentiation is apparent within the successive lymph gland lobes along the anteroposterior axis: the anteriormost lobes contained numerous proPO-positive cells, whereas the adjacent, more posterior lobes contain no or few positive cells and the most posterior lobes are totally devoid of differentiating crystal cells. In Nts1 mutants placed at a restrictive temperature, the number of proPO-positive cells is significantly reduced in the lymph glands, and they were occasionally totally absent. A similar phenotype was observed in a Su(H) and in a Ser mutant context. Conversely, activation of the Notch pathway in NMcd8 larvae and in larvae carrying UAS-Nic, UAS-Ser, or UAS-Dl transgenes driven by hsp-Gal4 dramatically increased the number of proPO-positive cells in lymph glands. Not only are the anteriormost lobes packed with such cells, but more posterior lobes are often found to contain large numbers of differentiating crystal cells. As expected, similar phenotypes are obtained when the e33C-Gal4 driver is used; this driver is strongly expressed in the lymph glands (Duvic, 2002).
Dorsoventral (DV) patterning is crucial for eye development in
invertebrates and higher animals. DV lineage restriction is the primary event
in undifferentiated early eye primordia of Drosophila. In
Drosophila eye disc, a dorsal-specific GATA family transcription
factor pannier (pnr) controls Iroquois-Complex
(Iro-C) genes to establish the dorsal eye fate whereas Lobe
(L), which is involved in controlling a Notch ligand Serrate
(Ser), is specifically required for ventral growth. However, fate of
eye disc cells before the onset of dorsal expression of pnr and
Iro-C is not known. L/Ser have been shown to be expressed in entire early
eye disc before the expression of pnr and Iro-C is initiated
in late first instar dorsal eye margin cells. The evidence suggests that
during embryogenesis pnr activity is not essential for eye
development. Evidence that loss of L or Ser
function prior to initiation of pnr expression results in elimination
of the entire eye, whereas after the onset of pnr expression it
results only in preferential loss of the ventral half of the eye. Dorsal eye disc cells also become L or Ser dependent when
they are ventralized by removal of pnr or Iro-C gene
function. Therefore, it is proposed that early state of the eye prior to DV
lineage restriction is equivalent to the ventral half and requires L and
Ser gene function (Singh, 2003).
Previously, L/Ser were thought to be required for ventral eye growth after the DV lineage
restriction boundary was established, which corresponds to the onset of
expression of dorsal eye selectors. The results, however, clearly suggest that
L/Ser are required much earlier for the growth of the entire early
eye disc, even before the DV patterning is established. In contrast to the
function of dorsal selector genes in eye patterning, L and
Ser have been shown to play a distinct role in controlling
ventral-specific growth of eye disc (Singh, 2003).
Loss-of-function phenotypes of L or
Ser are restricted to the ventral eye. The spatial as well as temporal requirement of these genes in the
ventral eye pattern formation were examined. Extent of loss of ventral eye
pattern in loss-of-function clones of L/Ser varies along the temporal
scale. During early eye disc development, prior to onset of pnr
expression in dorsal eye, removal of L or Ser function
results in complete elimination of the eye field, whereas later when dorsal
eye selector genes starts expressing the eye suppression phenotype becomes
restricted only to the ventral eye. Therefore, DV lineage
border in the eye can also be interpreted as the border between the cells
sensitive and insensitive to the L/Ser gene function (Singh, 2003).
The eye antennal disc has the most complex origin in the embryo. The eye
disc is initiated from a small group of ~70 precursor cells on each side
contributed by six different head segments of the embryo. These embryonic precursors do not physically separate from
the surrounding larval primordia and are therefore difficult to discern
morphologically (Singh, 2003 and references therein).
Once the cells for the eye-antennal disc are committed, these discs
proliferate and undergo differentiation into an adult eye, which requires
generation of DV lineage restriction in eye. There are possibly three
different ways by which genesis of DV lineage in the eye can be explained.
Early first instar larval eye disc may initiate either from only dorsal, only
ventral or from both DV lineages. Based on results from studies of
expression patterns and analysis of mutant phenotypes, it is proposed that larval eye primordium initially comprises only the ventral-equivalent state
rather than well-defined DV or dorsal states alone. The initial state of
eye is referred to as ventral equivalent state because, at this stage, dorsal and ventral identity is not yet generated. DV lineage restriction is established later
after the onset of pnr expression. The cells of the initial
ventral-equivalent state are similar to the ventral eye cells that are
generated after DV specification. The similarity is in terms of their
requirement of L/Ser for growth and maintenance, and the absence of
the dorsal selector expression. How dorsal lineage is initiated in the early
eye disc is not yet clear. Once the DV lineage restriction is established, N
signaling is initiated at the equator, a border between dorsal and ventral
compartments. Activation of N signaling promotes proliferation, which is
followed by differentiation of eye disc into adult compound eye (Singh, 2003).
The ventral-equivalent state model is supported by two observations: 1)
presence of Ser and L expression in the dorsal and ventral
eye disc of the early first instar larva and 2) change of dorsal eye fate to
ventral upon removal of dorsal selectors. The
mutants, which affect ventral eye development, show two major phenotypes in
eye: either there is no or very small eye, or there is a preferential loss of
ventral eye based on the time they affect their function but none of the
mutants for dorsal eye selectors show phenotypes of loss of only dorsal eye.
Conversely, loss-of-function clones of pnr or Iro-C causes
dorsal eye enlargement or ectopic eye formation rather than loss of only
dorsal eye clonal tissue. This phenotype is probably due to generation of
ectopic boundary of pnr-expressing and non-expressing cells (rather
than absence of pnr), which could be important for promoting eye
growth. Overexpression of Ush or Fog proteins in eye discs results
in loss of pnr activity, causing complete elimination of eye
development. By removing pnr activity at different time points
it was found that pnr activity in embryo and early first instar is not
essential for eye disc development. Later, pnr becomes essential for DV patterning consistent with its strong expression in dorsal margin of eye disc after the early first instar stage (Singh, 2003).
In contrast to enlargements or ectopic eyes induced by loss-of-function
clones of dorsal selectors, the loss-of-function clones of L or
Ser always resulted in the elimination of the eye fate.
L/Ser are primarily required for the maintenance and development of
ventral or ventral-equivalent state of the eye, whereas dorsal genes establish
the DV border. This suggests that dorsal genes and L/Ser, although
involved in a common goal of generation of DV lineage in eye, probably affect
eye development at two different tiers (Singh, 2003).
Fng, another essential component of DV patterning in eye, is expressed
preferentially in the ventral domain of early eye disc and is required for
restriction of N signaling to the DV border.
Although fng is known to act upstream of Ser in the wing and
eye discs,
there is also an apparent difference between the two genes. Unlike
L/Ser, the main function of fng seems to affect DV
ommatidial polarity but not the growth.
This suggests that fng may be selectively required for DV patterning
after dorsal selectors initiate domain specification. This may be the reason
why phenotypes of loss-of-function clones of fng are different from
those of L and Ser in the eye. It has been observed that the
pattern of fng expression is not altered in L mutants, and
vice versa, supporting the independent functions of these two genes in
controlling DV border formation and growth of ventral domain (Singh, 2003).
The function of Pnr in organizing the DV pattern from an initial
ventral-equivalent state raises an interesting question of whether similar
patterning processes occur in other developing tissues and organs.
Interestingly, Pnr is expressed in a broad dorsal domain in early embryos, but
later refined in a longitudinal dorsal domain extending along the thoracic and
abdominal segments. During this stage, Pnr has an instructive and
selector-like function, determining the identity of the medial dorsal
structures. It has been shown that loss of pnr eliminates the
dorsomedial pattern in the larval cuticle whereas the dorsolateral pattern
extends dorsally without cell loss. This suggests that DV pattern in the larval cuticle is established with the onset of Pnr expression in the dorsomedial domain, and ventral may be the initial fate of epidermal cells (Singh, 2003).
During the development of the Drosophila wing, the activity of the Notch signalling pathway is required to establish and maintain the organizing activity at the dorsoventral boundary (D/V boundary). At early stages, the activity of the pathway is restricted to a small stripe straddling the D/V boundary, and the establishment of this activity domain requires the secreted molecule Fringe (Fng). The activity domain will be established symmetrically at each side of the boundary between Fng-expressing and non-expressing cells. Evidence is presented that
the Drosophila tumor-suppressor gene lethal giant discs (lgd), a gene whose coding region has yet to be identified, is required to restrict the activity of Notch to the D/V boundary. In the absence of lgd function, the activity of Notch expands from its initial domain at the D/V boundary. This expansion requires the presence of at least one of the Notch ligands, which can activate Notch more efficiently in the mutants. The results further suggest that Lgd appears
to act as a general repressor of Notch activity, because it also affects vein, eye, and bristle development (Klein, 2003).
It has also been observed that wingless (wg) is expressed ectopically in
the pouch of lgd mutants during wing development. Similar phenotypes are observed,
if the Notch pathway is ectopically activated during wing
development, raising the possibility
that the lgd mutant phenotype could stem from the ectopic
activation of the Notch pathway. The Notch pathway is indeed ectopically
active in lgd mutants, and hyperactivation as well
as ectopic activation of the pathway accounts for the lgd
phenotype during wing development. In lgd mutants, the
expression of Notch target genes along the D/V boundary is
expanded, indicating that Lgd is required for the restriction
of Notch activity to the D/V boundary. Furthermore, the
mutant phenotype of lgd is suppressed by concomitant loss
of Presenilin or Suppressor of Hairless function, indicating that the mutant phenotype is caused by the
activation of the Notch pathway. Evidence is provided that the
activity of fng and Serrate seem to be dispensable in lgd mutant wing disc and that Delta can activate Notch efficiently enough to maintain its activity during wing development. The presented results indicate that the negative regulation of Notch
by Lgd is not restricted to wing development and occurs
during several other developmental processes, such as vein,
eye, and bristle development, suggesting that Lgd suppresses
the activity of the Notch pathway in a variety of developmental processes (Klein, 2003).
Loss of lgd function leads to an overgrowth of the imaginal
discs, clearly noticeable in the wing region of the wing
disc, which becomes enlarged and flat (Bryant, 1971). wg expression is normally restricted to the D/V boundary of the wing pouch. In lgd mutants, wg is activated ectopically in a
much broader domain that extends into the wing pouch. In addition, lgd
mutant wing discs often develop a second wing pouch in the
region of the anlage of the scutellum. Similar phenotypes are caused by gain-of-function
alleles of N (for example, Abruptex) and are also observed
upon expression of the activated intracellular form of
Notch, Nintra, or expression of Notch ligands, such as Dl. The ectopic activation of wg can already be observed in early third instar wing
discs and precedes the visible morphological changes that
occur at later stages. The deficiency Df(2L) FCK-20 deletes the lgd locus, allowing the classification of the relative strength of the available alleles. The
phenotype is always variable, but the overall phenotype of
lgdd7 and lgdd10 in homozygotes and in trans over Df(2L)FCK-20 is very similar, indicating
that these two alleles are strong, probably amorphic alleles.
lgdd4 and lgdd1 are weaker alleles. All alleles display a qualitatively similar phenotype over the deficiency as in
homozygotes, indicating that the observed phenotype is
probably caused by the loss-of-function of the lgd gene (Klein, 2003).
The similarity between the loss of lgd function and
ectopic N activation suggests that the phenotype of lgd
could be caused by ectopic activation of the Notch pathway.
To examine this possibility, the expression of
E(spl)m8, cut, Dl, and Ser was monitored as well as the activity of the
vg-boundary enhancer (vgBE) in mutant wing discs. The
expression of all these markers is initiated in cells at the
D/V boundary in a Notch-dependent manner. The vgBE is initially expressed along the D/V boundary of the wing, but late in the third instar, it is
activated in an additional stripe along the anteroposterior
compartment boundary (A/P boundary), which is also dependent
on Notch activity. Both domains depend on the presence of a single Su(H) binding site in the enhancer. Similarly, the expression of cut and E(spl)m8 is initiated in cells at the boundary by the Notch-pathway, and E(spl)m8 is also dependent
on the presence of Su(H) binding sites in its promoter. As described above, the expression of Dl and Ser is
more complex but always dependent on the activity of
Notch in cells at the D/V boundary. In lgd mutant wing
discs, the vgBE as well as cut, Dl, Ser, and E(spl)m8 are
activated ectopically within the wing pouch. The activation of the vgBE is dependent on the presence of the Su(H) binding site in the enhancer,
since a version lacking it shows no ectopic activity
in the mutants. As in the case of wg, the expression
of the vgBE is already expanded in early third larval
wing discs. Altogether, these results show
that the loss of lgd function leads to the ectopic expression
of Notch target genes. This suggests that the Notch pathway
is ectopically activated in lgd mutants (Klein, 2003).
All tested Notch-target genes are ectopically activated
in lgd mutant wing discs or lgd mutant cell clones.
The ectopic activation of Notch target genes as well as the
observed overproliferation of lgd mutants is abolished in
lgd;Psn double mutants. In addition, Notch target gene
expression is also abolished in Psn or Su(H) mutant clones
generated in lgd mutant wing imaginal discs. These data
suggest that the Notch pathway becomes ectopically active
in the absence of lgd function. Furthermore, the fact that Delta
alone seems to provide sufficient Notch activity to sustain
wing development in lgd mutants indicates that the pathway
can be activated more efficiently in the mutant background.
The activation of Notch is a consequence of
loss of lgd function also in other developmental processes,
such as bristle, leg, and wing vein development. Thus, the presented data make lgd a good candidate gene that regulates activity of the Notch pathway during adult development of Drosophila (Klein, 2003).
The phenotype of Ser;lgd double
mutant wing discs was further analyzed to examine the effect of loss of one Notch
ligand in lgd mutants. Loss of Ser function leads to the loss
of most of the wing blade and the margin. The
presence of a remnant of the wing pouch is due to the fact
that the Notch pathway is active during early stages of wing
development. This activation is achieved through a residual
expression of Dl. Animals
of the Ser;lgd double mutant phenotype develop very
slowly, and only few larva survive until the third instar. The
wing imaginal discs of the larva have expanded wing
pouches and, in contrast to Ser-mutant discs, they express
vg and Dl and wg in the wing blade. This shows, that in the absence of lgd function, the activity of Ser is not required to maintain
Notch-dependent gene activity. In summary, the observed
genetic interactions reveal a functional relationship between
the Notch and lgd locus and support the conclusion that lgd
is a negative regulator of the Notch pathway (Klein, 2003).
The observation that loss of lgd function can compensate
for the loss of Ser function raises the possibility that Notch
could be activated in a ligand-independent manner in the
absence of lgd function. To test this possibility,
Ser/Dl double mutant clones were generated in lgd-mutant wing discs. The
clones were induced through combining the Flp/FRT and
the targeted Gal4-System. In the experiments described
here, the expression of UASFlp was activated with sdGal4.
sdGal4 is active throughout wing development and therefore
activates UAS Flp expression at all stages of development (Klein, 2003).
In the clones, the expression of the Notch-regulated
genes wg and cut was interrupted in the center of the clone
area, suggesting that the expression
of these genes in lgd mutants depends on Notch ligands.
However, several interesting additional effects were observed.
(1) Surprisingly, wg and cut expression was induced
on both sides of the clone boundary, which can be
clearly seen in clones located outside the expanded expression
domain normally observed in lgd mutants . The effect is observed in the dorsal as well as the ventral half of the pouch. This suggests that the removal of the ligands leads to the activation of Notch at the boundary of Dl/Ser-expressing
and nonexpressing cells. (2) In several cases, the
expression of cut and wg expands outside the clone, even far
away from the clone boundary. This effect is
biased, and the expansion toward the D/V boundary is stronger (Klein, 2003).
(3) The expression of the Notch targets is activated
up to three-cell diameter into the clone in a graded manner. Since the ligands are membrane anchored and
thought to signal to adjacent cells, an activation of Notch
target gene expression beyond one-cell diameter into the
clone is not expected. One possibility is that the induction of
Cut by Notch is indirect and mediated by a diffusible factor
that is induced at the clone boundary (Klein, 2003).
However, it was found that clones of Su(H) mutant cells in lgd
mutant discs lose expression of Notch target genes, such as
Cut, indicating that the cells require a
functional Notch pathway to activate expression of its target
genes. Similar results were obtained with Psn mutant
clones, using Wg expression as a read out of Notch activity. These results rule out the possibility that the target genes of Notch are induced indirectly through a
diffusible factor induced by the Notch pathway (Klein, 2003).
In summary, these results suggest that, in lgd mutant
wing blades, all cells that express Notch-regulated genes
require the activity of the signal cascade and receive a signal
through Dl and/or Ser. In addition, they indicate that, in the
Ser;lgd double mutant wing discs described above, Dl
alone is sufficient not only to initiate, but also to maintain
N-activity during wing development. Hence, it seems that
Notch can be activated more efficiently by Dl in the absence of lgd (Klein, 2003).
Notch is a single-pass transmembrane receptor. The N signaling pathway is an evolutionarily conserved mechanism that controls various cell-specification processes. Drosophila Deltex (Dx), a RING-domain E3 ubiquitin ligase, binds to the N intracellular domain, promotes Nís endocytic trafficking to late endosomes, and has been proposed to activate Suppressor of Hairless [Su(H)]-independent N signaling. However, it has been difficult to evaluate the importance of dx, because no null mutant of a dx family gene has been available in any organism. This study reports the first null mutant allele of Drosophila dx. dx is involved only in the subsets of N signaling, but is not essential for it in any developmental context. A strong genetic interaction exists between dx and Su(H) suggested that dx might function in Su(H)-dependent N signaling. These epistatic analyses suggested that dx functions downstream of the ligands and upstream of activated Su(H). A novel dx activity has been uncovered that suppresses N signaling downstream of N (Fuwa, 2006).
This study shows that the ability of Dl/Ser ligands to activate N signaling is partially reduced in the wing discs of the dx152 mutant. This result suggests that dx acts downstream of the Dl/Ser ligands to activate N signaling. When Dx is overexpressed, N signaling is induced independent of the presence of the Dl/Ser ligands. It is therefore possible that artificially elevated levels of Dx somehow overcome the Ser/Dl requirement for N activation. However, it is notable that both Dl and Ser showed a substantial ability to stimulate N signaling in the dx152 mutant disc, but they failed to activate N signaling in Su(H) mutant clones. Therefore, both dx and Su(H) play roles in ectopic vgBE activation, but while Su(H) is indispensable, Dx is required only for strong signal induction (Fuwa, 2006).
Genetically, dx has been considered a positive regulator of N signaling in Drosophila. Furthermore, mammalian homologues of dx activate the reporter genes of N signaling target genes. Also, it was shown that Dx and a dominant-negative form of Nedd4 activate the E(spl)mγ promoter in Drosophila cultured cells. However, in contrast, a human Dx homolog antagonizes N signaling in cortical neurons. This discrepancy could be explained by the finding that dx both activates and suppresses N signaling (Fuwa, 2006).
In this study, a novel genetic interaction involving dx was uncovered. It has been reported that N shows a dominant lethal interaction with hypomorphic alleles of dx. Here, it was found that Ser94c shows a dominant lethal interaction with dx152. This result was unexpected, because Dl, which functions more broadly than does Ser, did not show a lethal interaction. Thus, it is possible that dx and Ser have common tissue specificity. However, the developmental and molecular basis of this interaction between dx and Ser remains to be addressed (Fuwa, 2006).
Serrate and the establishment of leg segments (part 1/2) 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 Notch 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).
Serrate and the establishment of leg segments (part 2/2) Effects of mutation continued: part 2/2
Serrate:
Biological Overview
| Evolutionary Homologs
| Regulation
| Developmental Biology
| Effects of Mutation
| References
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