apterous
Hox genes control segment identity in the mesoderm as well
as in other tissues. Most evidence indicates that Hox genes
act cell-autonomously in muscle development, although this
remains a controversial issue. apterous
expression in the somatic mesoderm is under direct Hox
control. A small enhancer element
of apterous (apME680) has been identified that regulates reporter gene
expression in the LT1-4 muscle progenitors. The product of the Hox gene Antennapedia is present in the
somatic mesoderm of the second and third thoracic
segments. Through complementary alterations in the
Antennapedia protein and in its binding sites on apME680,
it has been shown that Antennapedia positively regulates apterous in
a direct manner, demonstrating unambiguously its cell-autonomous
role in muscle development. LT1-4 muscles contain more nuclei in the
thorax than in the abdomen and it is proposed that one of the
segmental differences under Hox control is the number of
myoblasts allocated to the formation of specific muscles in
different segments (Capovilla, 2001).
A fragment of 680 bp, located in the second largest ap intron, is capable of directing lacZ reporter expression starting from stage 10 in clusters of cells very similar to those expressing ap at this stage. This fragment is called apME680 (for ap-muscle-enhancer-680) because it directs muscle-specific reporter gene expression. At stage 13, beta-galactosidase is detected
in one continuous cluster in T2 and T3, while two smaller
clusters, located at the dorsal and ventral limits of the thoracic
clusters, are detected in segments A1-A7. In segment
A8, a unique smaller cluster is detected. These beta-galactosidase-positive cells contribute to the formation of muscles LT1-4 in segments T2-A7 and to muscle LT1 in A8. These are a subset of the muscles originating from ap-expressing
cells, since ap is expressed also in the progenitors of
muscles VA2 and VA3. Thoracic muscles LT1-4 are differ slightly from the same
abdominal muscles. In particular, muscle LT4 extends more dorsally and ventrally in the thorax than in the abdomen (Capovilla, 2001).
The question of the significance of the homeotic regulation of ap by Antp was addressed. The perdurance of beta-galactosidase allows the labeling of
thoracic and abdominal LT1-4 mature muscles originating
from the cells expressing ap starting from the early germ band
extended stage. LT1-4 muscles present different
characteristics in the thorax and in the abdomen. In the thorax,
they contain more beta-galactosidase, they are more tightly
packed and, at least in the case of muscle LT4, extend more
dorsally and ventrally. These differences may be a consequence of more myoblasts contributing to the thoracic muscles than to the corresponding abdominal muscles. To
investigate this hypothesis, double labeling
experiments were performed using anti-beta-galactosidase to label muscles LT1-
4 and anti-MEF2 antibodies, which label all muscle nuclei. In wild-type embryos,
LT1-4 thoracic muscles do contain more MEF2-positive nuclei
than the same abdominal muscles. The number of nuclei was
compared in the T2, T3 and A1 hemisegments of ten
independent embryos. This quantitative analysis shows that, on
average, T3 muscles contain a total of 28 nuclei, while A1
muscles contain 19 nuclei. This difference is statistically significant. No significant differences were observed between the number of nuclei in T2 and T3. Consistently, highly packed nuclei are present in the medial portion of T2 and T3 muscles, but are absent in the same region of abdominal muscles (Capovilla, 2001).
The Wingless protein, in a role surprisingly distinct from its embryonic
segment polarity function, appears to be the earliest-acting member of the hierarchy of regulatory genes that subdivide the wing disc into discrete subregions. Wingless is crucial for
distinguishing the notum/wing subfields, and for the compartmentalization of the dorsal and ventral
wing surfaces. wingless signaling is required to restrict the expression of the apterous gene to
dorsal cells and to promote the expression of the vestigial and scalloped genes that demarcate the
wing primordia and act in concert to promote morphogenesis (Williams, 1993).
Cell fate decisions in the early Drosophila wing disc assign cells to compartments (anterior or posterior and dorsal or ventral) and
distinguish the future wing from the body wall (notum). Egf receptor signaling stimulated by its ligand,
Vein, has a fundamental role in regulating two of these cell fate choices: (1) Vn/EGFR signaling directs cells to become notum by
antagonizing wing development and by activating notum-specifying genes; (2) Vn/EGFR signaling directs cells to become part of the
dorsal compartment by induction of apterous, the dorsal selector gene, and consequently also controls wing development, which
depends on an interaction between dorsal and ventral cells (Wang, 2000).
To determine when Vn/EGFR signaling is required for notum
development, the temperature-sensitive alleles,
Egfrtsla and vntsWB240 were used.
Inactivating Vn/Egfr activity during the second instar (a 24 hr period)
causes loss of the notum. The wing develops but
shows pattern abnormalities characteristic of vn hypomorphs. Later shifts during the third instar does
not cause loss of the notum. This demonstrates that Vn/Egfr
activity is required for notum development in the second instar when
wg is required to specify the wing. Thus, Vn
and Wg appear to have complementary roles and this
relationship has been examined by following their expression in mutants (Wang, 2000).
In second instar wild-type wing discs, wg is expressed
distally in a wedge of anterior ventral cells and vn is expressed
proximally. In vn null mutants,
the initiation of wg expression is normal as is expression
of its target gene optomotor-blind (omb). In wg mutants,
however, there is a dramatic and early expansion of vn
expression to include distal cells, presaging the development
of these cells as an extra notum. Together these results suggest that Vn has an early role in
establishing the notum and that Wg signaling is required to define a
distal domain that is reduced in Egfr activity to allow wing development (Wang, 2000).
To test the role of Vn/Egfr signaling in specifying notum an
examination was carried out to see whether the Iroquois complex (Iro-C) genes, ara and cap are targets of the pathway.
The Iro-C genes have been implicated in specifying notum cell fate because loss of function causes a transformation of notum to hinge. Furthermore, misexpression of
ara causes loss of the wing and a duplication of notum. Ectopic expression of an activated form of the receptor, Egfrlambdatop4.2 greatly
reduces the size of the wing and a small ectopic notum forms. vn is expressed in the presumptive notum in early second
instar discs and Caup/Ara are expressed in the presumptive
notum at the end of the second instar. In early third instar wing discs, Caup/Ara are
expressed in a domain that overlaps with vn. In
vn mutants, this expression of Caup/Ara is lost and
loss of Egfr signaling, in Egfrts clones, in the
medial notum results in a loss of Caup/Ara expression.
However, clones in the lateral notum continued to express Caup/Ara, suggesting other factors regulate Iro-C gene expression in these cells at this stage (Wang, 2000).
Activation of Iro-C genes could account for the requirement for Egfr
activity to specify the notum at the end of the second instar as this
correlates with when these genes are first expressed. However, loss of
Egfr signaling at a slightly earlier time (mid-first instar to
mid-second instar, see below), prior to activation of the Iro-C genes,
also results in loss of the notum. A possible explanation for this
comes from the finding that vn expression is lost in
vn mutants. This suggests Egfr activity must be
sustained, via a positive feedback loop involving transcriptional activation of vn, during the second instar, to activate the
Iro-C genes and hence specify notum at the end of this period.
Interestingly, the vn gene is also a target of Egfr signaling
in the embryo (Wang, 2000 and references therein).
It is suggested that the mechanisms by which wg and vn
specify alternate cell fates in the early wing disc, wing, or notum are
antagonistic. This is based on the observation that loss of Wg results
in the spread of vn expression and the supposition that the
resulting ectopic Egfr activity causes loss of the wing and a double
notum phenotype. Further evidence that Vn/Egfr signaling represses wing development comes from the results of misexpressing a constitutive receptor, Egfrlambdatop4.2, in the presumptive
wing. In these flies, the wing is reduced to a stump covered with
sensilla characteristic of the proximal wing (hinge) region and
expression of the wing specific gene vestigial (vg) is repressed. Ectopic notal structures also form from the ventral pleura. The ability of ectopic Egfr signaling to suppress wing development is cell
autonomous because clones of cells expressing Egfrlambdatop4.2
lack vg expression. In adult wings these clones
produced outgrowths lacking wing characteristics but are otherwise
difficult to characterize (Wang, 2000).
Although vn expression expands in wg mutants, no reciprocal spread of wg expression was observed in
vn mutants that would have been indicative of a double wing
phenotype. However, when Vn/Egfr signaling is inhibited in
the notum by expressing a ligand antagonist (Vn::Aos-EGF) under the control of ptc-Gal4, ectopic wings are
induced in ~10% of the flies. This result demonstrates
that presumptive notal tissue can be transformed to wing by reducing
Egfr signaling. However, the transformation occurs only when Egfr
signaling is reduced in a subset of cells, rather than all cells in
the notum (as in a vn mutant). This may reflect the indirect requirement for Egfr activity to also promote wing development (Wang, 2000).
The loss of notum phenotype is characteristic of vn
hypomorphs but in null vn alleles and some Egfr
alleles both the wing and notum primordia fail to develop and the wing
discs remain tiny. Thus, although ectopic activity of Egfr in the distal disc
represses wing development, the pathway is nevertheless normally
required for wing development. Using the temperature-sensitive
Egfrtsla allele it was found that this requirement is
restricted to the period from mid-first to mid-second instar. Key genes involved in wing development that are active at this time include wg and apterous
(ap). ap is expressed in dorsal cells and acts as a selector gene
to divide the disc into dorsal and ventral compartments. Regulation of Notch ligands by Ap
leads to Notch signaling at the DV boundary and the formation of an
organizer for wing outgrowth and expression of the wing-specific transcription factor vg (Wang, 2000 and references therein).
Of these two candidates, wg and ap, it seemed
unlikely that wg was the key gene affected by Egfr signaling
from mid-first to mid-second instar because wg expression is
normal in vn mutants at mid-second instar.
However, later in the second instar, wg expression normally
expands to fill the growing wing pouch and it was noted that in vn
mutants, wg expression fails to undergo this expansion. A similar defect in wg expression is seen in ap
mutants consistent with Ap function being impaired
in vn mutants. Remarkably, ap expression is
completely absent in second instar vn mutant discs. Thus, loss of Ap can explain why there is no wing in vn
mutants. This is supported by the demonstration that ectopic
ap is capable of rescuing wing development in vn
mutants (Wang, 2000).
Several additional lines of evidence demonstrate that ap is a
cell autonomous target of Vn/Egfr signaling and that this relationship exists only transiently in early wing development: (1) ap
expression partially overlaps that of vn in the second instar; (2) ap can be induced ectopically in ventral
clones misexpressing an activated form of the receptor,
Egfrlambdatop4.2; (3) Egfrtsla
mutant clones generated in the first instar show autonomous loss of
ap expression, whereas clones generated in the
second instar express ap normally. Finally,
loss of Egfr activity in whole discs from mid-first to mid-second
instar results in complete loss of ap expression, whereas
ap is still expressed in discs from larvae given a temperature
shift slightly later during the second instar (Wang, 2000).
The results described here suggest that division of the early wing
disc into presumptive wing and body wall regions is defined by the
action of two secreted signaling molecules, Wg and Vn. wg, a pro-wing gene, is required to
repress vn expression, which at high levels antagonizes wing
development. Antagonism between Wg and Egfr signaling has also been
demonstrated in segmental patterning of the embryo and in development of the head and third
instar wing pouch, suggesting
such a relationship between these pathways may be a common theme in a
number of cell fate choices. Finding that one of the main functions of
Wg in early wing specification is to repress Vn/Egfr signaling in the distal region of the early disc raises the question as to whether this
is the only role of Wg in wing specification and hence if wing-cell
fate can be specified in the absence of both signals. This seems
unlikely, because nubbin, an early wing cell marker, is not misexpressed proximally in a vn mutant, where cells would lack both signals (Wang, 2000).
Vn/Egfr signaling promotes development of the notum by maintaining its
own activity through transcriptional activation of vn itself,
and also promotes expression of ap. Thus, both vn and ap appear to be targets of Egfr signaling, but the domain of
ap is clearly wider than that of vn, indicating that
ap can be activated at a lower signaling threshold than
vn. Vn is a secreted molecule and thus could generate a
gradient of Egfr activity. This provides an explanation for how Egfr
signaling can regulate both wing and notum development: vn
autoregulation and notum development requires high Egfr signaling
activity while ap expression and subsequent wing development
requires lower signaling activity (Wang, 2000).
Interestingly, vertebrate Egfr and its ligands are expressed in the
chick limb bud in a pattern that appears to overlap with the vertebrate
ap homolog Lhx2, and these factors are required for
limb outgrowth in the chick. In light of the present results it will be important to
determine whether Egfr signaling controls Lhx2 expression and thus plays a role in regulating outgrowth of the vertebrate limb. These
results may also have implications for the evolution of insect wings.
If the control of body wall development by Egfr signaling is ancestral,
and comparative analysis of other arthropods will be required to assert
this, then one of the first steps towards evolution of wings could have
occurred when Egfr signaling assumed control of ap (Wang, 2000).
Growth and patterning of the Drosophila wing imaginal disc depends on its subdivision into dorsoventral (DV)
compartments and limb (wing) and body wall (notum) primordia. Evidence is presented that both the DV and
wing-notum subdivisions are specified by activation of the Drosophila Epidermal growth factor receptor (Egfr). Egfr signaling is necessary and sufficient to activate apterous (ap) expression, thereby segregating
the wing disc into D (ap-ON) and V (ap-OFF) compartments. Similarly, Egfr signaling directs
the expression of Iroquois Complex (Iro-C) genes in prospective notum cells, rendering them distinct from, and immiscible with, neighboring wing
cells. However, Egfr signaling acts only early in development to heritably activate ap, whereas it is required persistently during subsequent
development to maintain Iro-C gene expression. Hence, as the disc grows, the DV compartment boundary can shift ventrally, beyond the range of the
instructive Egfr signal(s), in contrast to the notum-wing boundary, which continues to be defined by Egfr input (Zecca, 2002b).
The subdivision of the wing imaginal disc into AP and DV compartments, as well as prospective body wall (notum) and limb (wing) territories is marked by the expression of particular regulatory genes, such as the selector gene engrailed (en) in the P compartment, the selector gene apterous (ap) in the D compartment, and the genes of the Iroquois Complex (Iro-C) [mirror (mirr), auracan (ara) and caupolican (caup)] in the lateral notum. In mature third instar wing discs, the Iro-C genes are expressed not only within the prospective lateral notum, but in additional locations, including a thin stripe of cells that extends ventrally along the edge of the disc, as well as in specific subpopulations of cells in the prospective wing blade. This study addresses the role of Egfr signaling in controlling notum development and Iro-C gene expression therein, and then focuses on the role of Egfr signaling in inducing ap expression and establishing the DV compartments (Zecca, 2002b).
Egfr/Ras signaling is both necessary and sufficient to activate ap expression in early wing disc cells. Furthermore, evidence is provided that each wing disc cell chooses to express, or not to express, ap at this time, depending on its level of Egfr/Ras activation. However, in contrast to the Iro-C genes, the descendents of each cell then inherit this initial choice without further reference to Egfr/Ras signaling. The results of eliminating Egfr/Ras activity before the establishment of the DV compartments are particularly striking. Early loss of Egfr activity causes dorsally positioned cells within the disc to choose, incorrectly, to become V compartment founders. These cells and their descendents generally sort into the existing V compartment or out of the disc epithelium. In rare cases, they can form an ectopic V compartment within the D compartment. By contrast, later loss of Egfr activity has no effect on the DV compartmental segregation. These findings establish that Egfr signaling is responsible for establishing the D and V compartments through the heritable activation of ap (Zecca, 2002b).
Although the Iro-C and ap genes are activated in overlapping dorsoproximal sectors of the early wing disc, the domain of ap expression expands relative to that of Iro-C gene expression during subsequent development, causing the DV boundary to be positioned up to 30 cell diameters ventral to the notum-wing boundary. It is suggested that this shift occurs because ap-expressing cells no longer depend on Egfr/Ras input to continue to express ap. Hence, as ap-expressing cells within the notum primordium proliferate, some will move out of range of the instructive Egfr ligand, cease to express Iro-C genes and enter the wing primordium. In the accompanying paper (Zecca and Struhl, 2002b), evidence is provided that this shift must occur in order for D and V compartment cells to interact to induce Wg and stimulate wing growth and differentiation (Zecca, 2002b).
These results raise intriguing questions about the mechanism of ap activation. For example, Egfr signaling induces ap expression only during a discrete window of opportunity during the second larval instar, even though Egfr signaling both precedes the initial activation of ap and continues thereafter. What makes the ap gene responsive to Egfr signaling only during this early window of opportunity? In addition, the state of ap gene expression during this period, whether 'on' or 'off', is inherited for the remainder of development. How are both states of expression rendered heritable? It is possible that a temporal signal, such as a flux of a unique combination of hormones (for example, ecdysone and juvenile hormone) or the unique prior history of signaling events in the early wing disc, might prime the ap locus for activation by Egfr signaling during this period. The state of expression chosen during this period might then be maintained subsequently by mechanisms involving positive autoregulation (for the 'on' state) or heritable silencing mediated by the Polycomb Group proteins (for the 'off' state). However, there is little evidence at present to support these speculations and the actual mechanisms remain unknown (Zecca, 2002b).
The subdivision of the Drosophila wing imaginal disc into dorsoventral (DV) compartments and limb-body wall (wing-notum) primordia depends on Epidermal growth factor receptor (Egfr) signaling, which heritably activates
apterous (ap) in D compartment cells and maintains Iroquois Complex (Iro-C) gene expression in prospective notum cells. The source, identity and mode of action of the Egfr ligand(s) that specify these subdivisions has been examined. Of the three known ligands for the Drosophila Egfr, only Vein (Vn), but not Spitz or Gurken, is required for wing disc
development. Vn activity is required specifically in the dorsoproximal region of the wing disc for ap and Iro-C gene expression.
However, ectopic expression of Vn in other locations does not reorganize ap or Iro-C gene expression. Hence, Vn appears to play a permissive rather
than an instructive role in organizing the DV and wing-notum segregations, implying the existance of other localized factors that control where
Vn-Egfr signaling is effective. After ap is heritably activated, the level of Egfr activity declines in D compartment cells as they proliferate and
move ventrally, away from the source of the instructive ligand. Evidence is presented that this reduction is necessary for D and V compartment cells to
interact along the compartment boundary to induce signals, like Wingless (Wg), which organize the subsequent growth and differentiation of the wing
primordium (Zecca, 2002b).
All cells within the wing imaginal disc require a minimum level of Egfr/Ras activity to sustain a normal rate of proliferation. It is not known whether this activity reflects the basal activity of the Egfr/Ras transduction pathway, or the response of the receptor to a specific ligand. However, it is clear that this low level of Egfr/Ras activity does not require Vn dependent Egfr signaling, since it has been shown that ectopic expression of Ap in vn mutant discs can rescue growth and differentiation of the wing primordium. This result demonstrates that the absence of wing development in vn mutant discs is an indirect consequence of the failure to establish an apON-apOFF interface (Zecca, 2002b).
During normal development, the ap and Iro-C genes are initially activated in overlapping dorsoproximal domains in response to Egfr signaling, and hence, at this early stage, it appears that most or all D compartment cells are exposed to relatively high levels of Egfr/Ras signaling. Thereafter, as the wing disc grows, ventrally situated D compartment cells inherit the 'on' state of ap expression, even as they populate areas of the disc progressively farther from the domain of high Egfr/Ras signaling and sustained Iro-C expression. It is suggested that the progressive reduction of Egfr/Ras activity in these ventrally situated D cells enables them to interact with neighboring V compartment cells to induce Wg and Vg expression and stimulate growth of the wing primordium. By contrast, early induced clones of RasV12-expressing cells autonomously express ap and experience persistent high levels of Ras activation, as indicated by sustained expression of the Iro-C genes. As a consequence, the ectopic DV boundary cannot shift outside of the domain of high Egfr/Ras signaling. Cells flanking this ectopic DV boundary fail to engage in the reciprocal induction of Wg and Vg expression or to stimulate growth. Hence, the apON-apOFF interface may normally have to shift to a region of relatively low Egfr activity for the DV boundary to acquire wing organizer activity (Zecca, 2002b).
The apON-apOFF interface may only be able to function as an organizer when cells on both sides are of prospective wing type. Prior to the initial activation of ap and the Iro-C genes, the nascent wing disc appears to be subdivided into mutually antagonistic domains of Egfr and Wg signaling that at least transiently define the incipient notum and wing primordia. Because ap and the Iro-C genes are initially activated in response to a common source of Egfr signaling, most or all D cells at this stage may be notum type. It is only later, when ventrally situated D cells move out of range of Vn-dependent Egfr signaling and switch to being wing type, that inductive interactions occur across the DV boundary to create a new and stable source of Wg signaling. It is suggested that cells on both sides of the DV boundary may have to be of wing type for the boundary to have organizer activity. One possible reason for why this might be the case is that vg, the selector-like gene that defines the wing state, is itself an integral component of the reciprocal signaling mechanism that allows D and V cells to induce the expression of DV boundary genes. High levels of Egfr/Ras signaling actively maintain Iro-C gene expression (and hence the notum state) and block vg expression. Hence, the DV boundary may normally have to shift ventrally, into a domain of low Egfr/Ras signaling and high Wg signaling that defines the incipient wing state, to allow the positive feedback loop of inductive signaling to initiate across the DV compartment boundary. Once this loop is established, it would provide a stable source of Wg and other signals generated along the DV boundary that govern the subsequent growth and differentiation of the wing blade (Zecca, 2002b).
Drosophila thoracic muscles are comprised of both direct flight muscles (DFMs) and indirect flight muscles (IFMs). The IFMs can be further subdivided into dorsolongitudinal muscles (DLMs) and dorsoventral muscles (DVMs). The correct patterning of each category of muscles requires the coordination of specific executive regulatory programs. DFM development requires key regulatory genes such as cut (ct) and apterous (ap), whereas IFM development requires vestigial (vg). Using a new vgnull mutant, a total absence of vg is shown to lead to DLM degeneration through an apoptotic process and to a total absence of DVMs in the adult. vg and scalloped (sd), the only known Vg transcriptional coactivator, are coexpressed during IFM development. Moreover, an ectopic expression of ct and ap, two markers of DFM development, is observed in developing IFMs of vgnull pupae. In addition, in vgnull adult flies, degenerating DLMs express twist (twi) ectopically. Evidence is provided that ap ectopic expression can induce per se ectopic twi expression and muscle degeneration. All these data seem to indicate that, in the absence of vg, the IFM developmental program switches into the DFM developmental program. Moreover, the muscle phenotype of vgnull flies can be rescued by using the activity of ap promoter to drive Vg expression. Thus, vg appears to be a key regulatory gene of IFM development (Bernard, 2003).
Thus the absence of Vg leads to IFM degeneration. Some IFM phenotypes have been reported for the vg83b27R allele, a strong allele of vg. In these flies, the DVMs are absent and some DLMs are missing. It has been shown that this phenotype is fully penetrant in vgnull flies and that apoptosis is involved in loss of IFMs. Since muscle attachment sites are normal in vgnull flies, the process of degeneration is different from that described in ap mutants. Phenotypic analysis shows that degeneration occurs during late metamorphosis (after 48 h APF) (Bernard, 2003).
In vgnull mutants all adepithelial cells express high levels of Ct, while this is normally only the case of DFM-forming myoblasts. Is DLM degeneration in vgnull mutants the result of engagement of DLMs toward a DFM-like differentiation process? To answer this question, ap expression was examined in vgnull developing and adult DLMs. In wild-type flight muscles, ap expression is specific to DFMs and begins at 17-19 h APF. In vgnull flies, ap expression is found in developing DLMs at 21 h APF, in myoblasts surrounding DLMs and in adult muscles. Moreover, an absence of actin 88F expression was found in vgnull developing IFMs, suggesting that IFM differentiation is disrupted. Interestingly, as in wild-type flies, no expression was found in adepithelial cells. These data show that ap starts to be expressed at the same stage in DLMs of vgnull flies and in DFMs of the wild type strain (Bernard, 2003).
In summary, the following has been demonstrated in vgnull flies: (1) DLM-forming myoblasts express high levels of Ct, an early marker for DFM-forming myoblasts and (2) myoblasts and developing and adult degenerating DLMs express ap, a specific late DFM marker, whereas actin 88F expression, an IFM-specific differentiation marker, is lost. According to these data, it is supposed that in the vgnull mutants, adepithelial cells and developing DLMs enter into a DFM-like development. The suggestion that ap ectopic expression may impose a DFM identity on the IFMs has already been proposed. However, an IFM-to-DFM transformation was not observed; rather, IFMs degenerated through an apoptotic process. Similarly, DFMs were not transformed into IFMs upon overexpression of Vg in DFM-forming myoblasts. Instead, DFM degeneration was obtained. This suggests that Vg and AP are major actors but are not sufficient for IFM and DFM development, respectively. Other signals and factors must be required to specify these muscles. Nerve-muscle interaction is associated with IFM development. Wnt oncogene analog 2 (Dwnt-2) expression is required in the vicinity of the developing DFMs for patterning of DFMs. Thus, it appears that adult muscle development requires complex interactions between several kinds of signals delivered in specific localizations. In vgnull homozygous flies, adepithelial cells and swarming myoblasts express DFM markers, but their position on the wing imaginal disc and in the pupa remains unchanged with respect to wild type. Thus, developing IFMs receive IFM signaling (at least nerve-muscle interactions), but myoblasts express apterous, a DFM maker. Moreover, they lack information necessary for formation of either DFMs or IFMs (absence of vg expression). It is suggested that IFM degeneration in vgnull homozygous flies is the result of this complex interaction between two contradictory signals (IFM and DFM) associated with incomplete signaling for formation of either type of muscle (Bernard, 2003).
Attempts were made to rescue the vgnull muscle phenotype by targeted Vg overexpression using the UAS-GAL4 system. Significant rescue was obtained with the ap-GAL4 driver. It is therefore likely that ectopic activation of the ap-GAL4 transgene in vgnull DLMs and myoblasts occurs when Vg is required for DLM formation. Since ap activation in vgnull myoblasts and developing DLMs occurs after puparium formation, it is concluded that a late Vg expression is sufficient to restore the DLM developmental process. This implies that adepithelial cell determination by the level of Ct at the wing disc is reversible. Thus, even though earlier Ct levels distinguish two adepithelial cell populations that will differentiate into DFMs or IFMs, definitive DFM versus IFM determination is a later event that takes place during metamorphosis. vg and ap could be key genes during specification of IFMs and DFMs, respectively. To support this hypothesis, ubiquitous overexpression of ap was shown to be sufficient to induce specific DLM degeneration. The way in which AP and Vg direct muscle development toward a DFM or IFM fate remains unclear. However, it is well known that muscle fibers express specific structural genes or isoforms. Since ap and vg encode transcription factors, they are probably involved in specific genes activation. For example, misexpression of ap in developing IFMs represses the expression of actin 88F, an IFM-specific actin gene. Moreover, no actin 88F expression is found in a vgnull context. However, it is not currently known whether AP or Vg can directly activate or repress structural genes. Interestingly, the Sd mammalian homolog (Transcription Enhancer Factor-1, TEF-1) has been shown to bind muscle-specific promoters, like the cardiac alpha-Myosin Heavy Chain and the cardiac Troponin T promoters. It is therefore possible that the Sd-Vg dimer plays a similar role in Drosophila, directly activating structural genes. Further studies are necessary to address this question (Bernard, 2003).
Thus DLMs degenerate by apoptosis in homozygous vgnull flies. This degeneration could be due to a misprogramming of myoblasts surrounding DLMs during development. The process that leads to apoptosis in these muscles remains to be determined. DLM degeneration is associated with an ectopic expression of Twi transcription factor. During flight muscle development, Twi expression is restricted to myoblasts and that persistent expression in developing muscles leads to muscle degeneration. Thus, Twi expression in vgnull mutants could be responsible for DLM degeneration. Finally, it has been shown that ectopic ap expression induces Twi expression in DLMs. Since AP and twi are known to be, respectively, activator and target of the N pathway, it can be hypothesized that AP activates Twi ectopically in vgnull DLMs through the N pathway. If this hypothesis is confirmed, it can be asked why AP does not activate Twi during normal DFM development. It is likely that numerous genes, other than vg and ap, are differentially activated during DFM and IFM development. Twi activation by AP could be repressed by one of these genes during DFM development (Bernard, 2003).
In this study, evidence is provided that vg is required to change DFM-forming myoblasts into IFM-forming myoblasts. As in wing development where Vg is considered as a selector gene, Vg could be a key gene in IFM specification. Its function would be equivalent to that of Ap for DFM development. DFM fate inhibition through repression of ct and ap by Vg seems therefore to be a key regulation feature of IFM development. Thus, correct programming and regulation of these three genes are necessary for correct patterning of Drosophila flight muscles (Bernard, 2003).
The distal region of the Drosophila leg, the tarsus, is divided into five segments (ta I-V) and terminates in the pretarsus, which is characterized by a pair of claws. Several homeobox genes are expressed in distinct regions of the tarsus, including aristaless (al) and lim1 in the pretarsus, Bar (B) in ta IV and V, and apterous (ap) in ta IV. This pattern is governed by regulatory interactions between these genes; for example, Al and Bar are mutually antagonistic, resulting in exclusion of Bar expression from the pretarsus. Although Al is necessary, it is not sufficient to repress Bar, indicating another factor is required. This factor has been identified as the product of the C15 gene, also termed clawless, a homeodomain protein that is a homolog of the human Hox11 oncogene. C15 is expressed in the same cells as al -- together, C15 and Al appear to directly repress Bar and possibly to activate Lim1. C15/Al also act indirectly to repress ap in ta V, i.e., in surrounding cells. To do this, C15/Al autonomously repress expression of the gene encoding the Notch ligand Delta (Dl) in the pretarsus, restricting Dl to ta V and creating a Dl+/Dl− border at the interface between ta V and the pretarsus. This results in upregulation of Notch signaling, which induces expression of the bowl gene, the product of which represses ap. Similar to aristaless, the maximal expression of C15 requires Lim1 and its co-factor, Chip. Bar attenuates aristaless and C15 expression through Lim1 repression. Aristaless and C15 proteins form a complex capable of binding to specific DNA targets, which cannot be well recognized solely by Aristaless or Clawless (Campbell, 2005; Kojima, 2005).
Bar expression is absent from the center of the leg, specifically from the cells expressing Al and C15. However, other genes, including ap and bab, are absent from a more extensive region in the center, and there is a gap between the C15 expression domain and Ap and Bab. Consequently, Ap expression is restricted to presumptive tarsal segment IV, where it overlaps with Bar. It has been suggested that, as well as activating genes such as al and Bar, EGFR signaling may directly repress genes in the center of the disc, possibly accounting for the absence of ap and bab in this location. Surprisingly, ap and bab expression, as well as Bar, is regulated by C15/Al. In both C15 and al mutant discs, Ap and Bab expression expands into the center of the disc. Consequently, in regard to Ap expression, the distal region of the leg adopts a tarsal segment IV-like fate. However, Nub, which is normally only expressed in ta V, is now co-expressed with Ap in the very center, indicating that the distal-most segment in C15 legs has characteristics of both ta IV and V (Campbell, 2005).
In wild-type discs, Ap expression is first detected slightly later than Bar, Al, or C15, but even at this time there is a clear gap between Ap expression and C15, indicating that C15/Al acts non-autonomously to repress ap. This is supported by two further studies: (1) unless there is a complete loss of C15 in homozygous mutant discs, Ap expression is not derepressed in C15 mutant clones in the center if the clones are not too large, indicating surrounding wild-type C15-expressing cells can rescue the mutant tissue; (2) ectopic expression of C15 results in non-autonomous repression of Ap (Campbell, 2005).
These results suggest that EGFR signaling represses gene expression in the center of the disc only indirectly through activation of C15/Al. This is also supported by two other observations. (1) Al is still expressed in C15 mutant discs, indicating that EGFR signaling levels are still very high in the center of these discs, but ap is not repressed (if ap is repressed directly by EGFR, its threshold for this would be lower than the threshold for activation of al because ap is repressed further from the source in the center than al is activated). (2) Ectopic expression of C15 results in non-autonomous repression of ap, but, if this is due to increased EGFR signaling in surrounding cells, then it should result in activation of EGFR targets such as Bar immediately adjacent to the cells expressing C15 (outside of the normal Bar domain), but does not. Consequently, it seems very likely that C15 uses an alternative mechanism to repress ap, most likely by upregulation of a signaling pathway in surrounding cells (i.e., ta V) (Campbell, 2005).
Examination of Bowl and Ap expression in leg discs reveals that there is a gap between their expression domains, even at a time when Ap expression is first detected in mid-third instars. This could indicate that Bowl acts non-autonomously to repress ap. However, the clonal analysis clearly shows that Bowl acts autonomously: any wild-type cells expressing Bowl has no influence on Ap expression in surrounding mutant tissue. It is possible that there is low-level Bowl expression in the 'gap' that cannot be detected with antibody staining. Another possible explanation is one of timing, and that Bowl is expressed in the cells in the 'gap' slightly earlier and that this is sufficient to silence the ap gene even before its expression can be detected more proximally. The possibility that bowl is expressed transiently in cells has been proposed to explain the observation that bowl mutant clones have effects in central regions of tarsus, i.e., in regions where its expression cannot be detected later (Campbell, 2005).
Thus, Bowl is required to repress ap expression in tarsal segment V and this predicts that C15 regulates bowl expression. This was confirmed by analysis of C15 mutant discs, in which Bowl expression in the center is lost, although other, more proximal, domains of expression are normal. The ring of Bowl in the distal tarsus is usually just two cells in width with the inner cell overlapping with C15, but the outer cell being outside the C15 domain, suggesting C15 can induce bowl non-autonomously. This is supported by the ability of cells ectopically expressing C15 to activate Bowl expression in surrounding cells. This ability is fairly limited, but would be expected because the endogenous C15-expressing cells only appear able to induce bowl in their immediate neighbor (resulting in a ring of bowl expression in a single row of cells surrounding the C15 domain (Campbell, 2005).
The transcription factor Suppressor of Hairless [Su(H)] belongs to the CSL transcription factor family, the main transcriptional effector of the Notch-signaling pathway. Su(H) is the only family member in the Drosophila genome and should therefore be the main transcriptional effector of the Notch pathway in this species. Despite this fact, in many developmental situations, the phenotype caused by loss of function of Su(H) is too weak for a factor that is supposed to mediate most or all aspects of Notch signaling. One example is the Su(H) mutant phenotype during the development of the wing, that is weaker in comparison to other genes required for Notch signaling. Another example is the complete absence of a phenotype upon loss of Su(H) function during the formation of the dorsoventral (D/V) compartment boundary, although the Notch pathway is required for this process. Recent work has shown that Su(H)/CBF1 has a second function as a transcriptional repressor, in the absence of the activity of the Notch pathway. As a repressor, Su(H) acts in a complex together with Hairless (H), which acts as a bridge to recruit the co-repressors Groucho and CtBP, and acts in a Notch-independent manner to prevent the transcription of target genes. This raises the possibility that a de-repression of target genes can occur in the case of loss if function of Su(H). This study shows that the weak phenotype of Su(H) mutants during wing development and the absence of a phenotype during formation of the D/V compartment boundary are caused by the concomitant loss of the Notch-independent repressor function. This loss of the repressor function of Su(H) results in a de-repression of expression of target genes to a different degree in each process. Loss of Su(H) function during wing development results in a transient de-repression of expression of the selector gene vestigial (vg). This residual expression of vg is responsible for the weaker mutant phenotype of Su(H) in the wing. During the formation of the D/V compartment boundary, de-repression of target genes seems to be sufficiently strong, to compensate for the loss of Su(H) activity. Thus, de-repression of its target genes obscures the involvement of Su(H) in this process. Furthermore, evidence that is provided Dx does not signal in a Su(H)-independent manner as has been suggested previously (Koelzer, 2006).
This work provides an answer to the observation that the patterning defects of Su(H) mutant wing imaginal discs is weaker than anticipated for a gene that encodes a factor that mediates most of the transcriptional activity of the Notch-signaling pathway. Su(H) is required for the formation of the D/V compartment boundary despite any obvious defect in this process in the absence of its function. In both processes, the explanation for the phenotype of Su(H) mutants is the loss of its function as repressor of transcription along with its function as an activator (Koelzer, 2006).
Loss of function of Su(H) leads to an arrest in the development of the sensory organ precursor cell of the bristle sense organ. Although it was possible to demonstrate genetically that de-repression of expression of some genes of the Enhancer of split-complex are responsible for the arrest, it was not possible to detect the expression of any of these genes directly. This work shows that de-repression of vg is a consequence of loss of Su(H) function during wing development. Although this de-repression is weak and transient, it is sufficient to establish more distal elements than in mutants of other genes necessary for Notch signaling. The results are in agreement with two reports that show de-repression of target genes in Su(H) mutants in other developmental processes such as mesectoderm specification and bristle development. Thus, de-repression of target genes appears to be a common phenomenon during Drosophila development, if Su(H) function is lost. Importantly, this de-repression can even become strong enough to obscure an involvement of Su(H) in a developmental process, the formation of the D/V compartment boundary. De-repression of target genes upon loss of the repressor function of Su(H) is an attractive explanation for the paradox that loss of Notch function during the first larval instar stage is cell lethal, but loss of Su(H) function is not. Presumably, the de-repression of expression of target genes that are required for cell survival guarantees the survival of Su(H) mutant cells. In contrast, a similar de-repression cannot occur in Notch mutant cells, and the cells undergo apoptosis. Although the repressor function has been initially found in cell culture experiments with the vertebrate ortholog CBF1, reports analyzing the consequences of loss of its repressor function during vertebrate development are missing. The presented results should encourage researchers to search for such an effect in their vertebrate model systems (Koelzer, 2006).
The results have important implications on the use of various mutants in order to analyze the function of the Notch pathway in a particular developmental process. They show that the phenotype of loss of function of Su(H), or its vertebrate ortholog CBF1, is not necessarily identical to that of loss of the Notch-signaling activity. It is possible that de-repression of Notch target genes occurs upon loss of function of Su(H) but not upon inactivation of the pathway by other means. Previous work indicates that only a subset of genes might be de-repressed in a developmental process if Su(H) is absent. For example, de-repression of expression of wg along the D/V compartment boundary has never been observed upon loss of Su(H) function. The de-repression of only a subset of target genes could cause a phenotype that is difficult to interpret. Thus, it is better to use alleles of genes such as Psn, kuz or nic, which do not affect the repressor function of Su(H), to determine the function of the Notch pathway within a process of interest (Koelzer, 2006).
The weaker phenotype of Su(H) mutants during wing development was considered an argument for the existence of a Su(H)-independent mechanism of Notch signal transduction. The current findings strongly argue against the existence of such a mechanism in the analyzed processes. Evidence has been provided for the existence of a Su(H)-independent Notch-signaling pathway that is mediated by Dx. Since the existence of such a pathway has been excluded in the two other situations, it was of interest to discover whether an alternative explanation might exist for observations on the role of Dx. Indeed no evidence was found that Dx participates in a Su(H)-independent Notch signal during wing development. The results suggest that in this case, the confusion came from analyzing a domain of the vgBE (domain 2) that appears not to be completely dependent on the function of Su(H). Using the MARCM technique to generate Dx expressing Su(H) mutant cell clones, it was clearly show that Dx depends on the function of Su(H) to induce target gene expression in ectopic places as well as along the D/V boundary. Thus, the results abolish three arguments for the existence of a Su(H)-independent signal transduction mechanism during wing development. However, this does not imply that such a pathway does not exist. Indeed, evidence exists that during dorsal closure of the embryo, Notch acts independently of Su(H), through the JNK pathway (Koelzer, 2006).
Recent work indicates that cell-cell interactions are required for the establishment of both the A/P as well as the D/V compartment boundaries. While it is clear that a transcriptional response mediated by the transcription factor Cubitus interruptus (Ci) is necessary to establish the A/P boundary, the situation at the D/V boundary was unclear. The possibility has been raised of a Su(H)-independent mechanism that is used to establish the D/V boundary. This mechanism might not even require a transcriptional response to the Notch signal. The results demonstrate that this is not the case: similar to the formation of the A/P boundary compartment boundary, a transcriptional response to the Notch signal is required for the segregation of dorsal and ventral cells, and this response is mediated by Su(H). Similar to Ci, Su(H) acts as a transcriptional activator at the D/V boundary, where Notch is active and as a transcriptional repressor in a complex with H, and probably Groucho and dCtBP away from the boundary. The results suggest that the loss of this repressor function results in the de-repression of the relevant target genes in a manner sufficient to allow the formation of the D/V compartment boundary even in absence of Su(H). Overall the scenario at the D/V boundary seems to be very similar to that proposed for the formation of the A/P compartment boundary. In this situation, En endows the posterior fate and regulates the expression of Hedgehog that signals to anterior cells. As a response to Hh, the transcription factor Ci is transformed from a repressor to an activator of transcription and activates the expression of target genes in a stripe along the anterior side of the A/P boundary. The results suggest a similar scenario for the formation of the D/V compartment boundary: similar to En, Ap imposes the dorsal fates on cells and activates the expression of Ser. Ser signals to the ventral cells at the D/V boundary. Similar to Hh transforming Ci from a repressor into an activator of transcription, Ser induced activation of the Notch pathway transforms Su(H) from a repressor into an activator. In analogy to En, it was found that Ap has a second, Notch-independent function during D/V boundary formation. As in the case for En, an attractive possibility is that Ap acts to repress activation of the relevant target genes of Su(H) in dorsal cells. This repression creates a strong difference in expression of these genes at the D/V boundary and eventually leads to a strong difference in adhesion between the dorsal and ventral cells. This repressor function of Ap would also explain why the compartment boundary can form in the absence of Su(H) function, since the de-repression of target genes of Su(H) would be still restricted to ventral cells leading to a similar, albeit weaker difference in expression of these genes and in adhesion at the D/V boundary. Furthermore, it explains why the formation of the boundary fails in the absence of the function of ap and Su(H), since in this case no strong difference in expression of target genes will be created (Koelzer, 2006).
It appears that very similar strategies are exploited at both compartment boundaries to achieve segregation of the cell lineages. However, in each situation, a set of different but mechanistically similar acting signaling molecules are used to achieve the segregation of cell populations and formation of a compartment boundary (Koelzer, 2006).
alpha PS1 integrin is expressed at high levels in the dorsal domain of the third instar wing disc, while PS2 integrin is restricted to the ventral epithelium. Ectopic ventral expression of ap induces ectopic ventral alpha PS1 integrin (Blair, 1994).
apterous also regulates two other genes: fringe and Serrate. Each have distinct roles in a novel cell recognition and signal induction process. FNG serves as a boundary-determining molecule such that Ser is induced wherever cells expressing fng and cells not expressing fng are juxtaposed. SER in turn triggers the expression of genes involved in wing growth and patterning on both sides of the DV boundary. Fringe induces Serrate by a apterous independent mechanism. Serrate, through interaction with Notch induces vestigial and wingless. The expression of wingless is induced through Notch is independent of the earlier expression of wingless involved in inducing dorsal apterous expression (Williams, 1993 and Kim, 1995).
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).
In the ventral ganglion, apterous is expressed in up to ten of the
~350 neurons in each hemi-segment. In each of six thoracic hemisegments and in the third
subesophageal hemisegments, the ap neurons include a
ventrolateral cluster of four or five cells. Double-labeling
with antiserum to the FMRFalpha propeptide shows that
one of the neurons in each cluster is the Tv neuron, a neuron committed to neuropeptide production. Double-labeled Tv neurons shows cytoplasmic
FMRFalpha immunoreactivity and nuclear beta-galactosidase
immunoreactivity, marking cells expressing Gal4 under the direction of an apterous promoter. The ap gene is also expressed in
several brain cells, one of which is the FMRFalpha-positive SP2
neuron. Other larval brain FMRFalpha neurons, such as
the neighboring SP1 neuron, do not express ap. The
restriction of ap and FMRFalpha co-expression to the Tv and SP2
neurons is constant throughout mature larval stages; in the
adult, additional neurons begin to express dFMRFalpha, and some of these, including the Tva and several
subesophageal neurons, also express ap (Benveniste, 1998).
Tv neurons first stain with antibodies to the
tetrapeptide FMRFalpha during stage 17.
FMRFalpha gene expression was measured using reporter expression driven be a
446 bp Tv neuron-specific enhancer sequence
located within the first kB of FMRFalpha 5'
flanking region. Ap is required for
normal initiation of neuropeptide expression by
the Tv neurons. Apterous is shown not to be required for the survival or
morphological differentiation of the Tv
neuron cluster. Apterous contributes to the initiation of
FMRFalpha expression in Tv neurons, but not in those
FMRFalpha neurons that do not express Apterous. Apterous
is not required for Tv neuron survival or morphological
differentiation. Apterous contributes to the maintenance
of FMRFalpha expression by postembryonic Tv neurons,
although the strength of its regulation is diminished.
Apterous regulation of FMRFalpha expression includes
direct mechanisms, although ectopic Apterous does not
induce ectopic FMRFalpha. These findings show that, for a
subset of neurons that share a common neurotransmitter
phenotype, the Apterous LIM homeoprotein helps define
neurotransmitter expression with very limited effects on
other aspects of differentiation (Benveniste, 1998).
The hypothesis that Ap regulates FMRFalpha in the Tv
neurons directly was tested by seeking potential Ap-binding sites within
the dFMRFa gene regulatory sequences. The search was confined
to the 446 bp Tv neuron-specific enhancer, which is highly
responsive to Ap levels and located in the 5' flanking
region. The 446 bp enhancer contains three sequences
corresponding to the six-nucleotide consensus binding site for
homeodomain proteins. All three of these sequences are shared between the
homologous regions of the FMRFalpha genes of two Drosophila
species: D. melanogaster and D. virilis. Electrophoretic mobility shift assays (EMSA) were used
to test the ability of Ap protein to bind in vitro to these three
sequences, as represented by three different 25 bp
oligonucleotide probes. Recombinant Ap homeodomain binds
all three oligonucleotide probes with different affinities, and at stoichiometries comparable to
those observed for other LIM homeoproteins binding in vitro. Ap binding
to these probes in vitro is sequence-specific: mutant
oligonucleotide probes, with clusters of 6-point mutations
replacing the TAATNN sequences do not bind Ap in these
assays.
It was then asked whether these Ap-binding sequences are
functionally important in vivo. Two mutant Tv-lacZ
constructs were used incorporating the same clustered point
mutations in the Ap-binding sequences used in the EMSA. In
first instar larvae, a construct containing mutations in Ap-binding
site C [(mC)Tv-lacZ ] shows slightly decreased
activity in Tv neurons and in ectopic cells relative to the wild-type
enhancer. Construct (mABC)Tv-lacZ, which
includes mutations in all three Ap-binding sequences, shows
no detectable activity in Tv neurons or ectopic cells.
These results show that at least two of the three elements within
the Tv neuron-specific enhancer that bind Ap in vitro are
critical for proper enhancer activity in vivo (Benveniste, 1998).
It is found that ap is expressed in more than 100 neurons in the
larval CNS, but that FMRFalpha is expressed in only eight of
these. Therefore, co-factors must be required to activate
FMRFalpha transcription in the Tv neurons or to repress FMRFalpha
transcription in other neurons that express Ap. Two lines of
evidence suggest that positively acting co-factors are required
for FMRFalpha gene activation by Ap. (1) Widespread ectopic
expression of Ap (ubiquitously or throughout the CNS) does not
induce ectopic FMRFalpha expression. (2) Ap expression in
embryonic Tv neurons begins soon after the birth of the cell and precedes dFMRFa expression by
at least 3-6 hours (Benveniste, 1998 and references).
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 has been 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).
Capricious and Tartan, two transmembrane proteins with leucine-rich repeats, contribute to formation of the affinity boundary between dorsal and ventral compartments during Drosophila wing
development. Engrailed/Invected expression confers posterior (P) identity and Apterous confers dorsal (D) identity in the wing disc. P compartment cells lacking engrailed/invected activity do not respect the anterior-posterior boundary. Likewise, dorsal cells lacking ap activity fail to respect the dorsal-ventral (DV) boundary in the wing disc. Modulation of Notch signaling has been implicated in DV boundary formation. Fringe acts as a glycosyltransferase to modify the receptor protein Notch in the dorsal compartment. Fringe activity makes D cells more sensitive to Delta, a ligand expressed by V cells and less sensitive to Serrate, the ligand expressed by D cells. Consequently, signaling by each ligand is limited to nearby cells on the opposite side of the boundary, with the result that high levels of Notch activity are limited to a narrow band of cells along the DV boundary. Although altering the signaling properties of cells by modulation of Fringe activity has been shown to allow cells to cross the boundary, Fringe activity has been shown to be insufficient to support boundary formation. This observation, together with the fact that Notch signaling is activated symmetrically has suggested that other Apterous-dependent cell interactions might be needed for formation of the DV affinity boundary. Evidence suggests that capricious and tartan are targets of Apterous that contribute to DV boundary formation in the wing disc. caps and tartan are expressed in the D compartment during boundary formation. Caps and Tartan confer affinity for D cells, assessed by sorting-out behavior. Caps supports boundary formation without conferring D signaling properties. Fringe, in contrast, confers dorsal signaling properties without affecting DV affinity. Thus, Caps, Tartan, and Fringe have complementary roles in boundary formation (Milán, 2001a).
Drosophila limbs develop from imaginal discs that are subdivided into compartments. Dorsal-ventral subdivision of the wing imaginal disc depends on apterous activity in dorsal cells. Apterous protein is expressed in dorsal cells and is responsible for (1) induction of a signaling center along the dorsal-ventral compartment boundary; (2) establishment of a lineage restriction boundary between compartments, and (3) specification of dorsal cell fate. The homeobox gene msh (muscle segment homeobox) acts downstream of apterous to confer dorsal identity in wing development (Milán, 2001b).
Four structural features distinguish the dorsal and ventral surfaces of the
adult wing: (1) bristle morphology in the anterior wing margin; (2)
the presence or absence of bristles in the alula; (3) the surface on which the veins are corrugated, and (4) the location of certain
sensory organs. The anterior wing margin (AWM) is composed of three rows of bristles, two located in the dorsal surface and one in the ventral. The dorsal wing margin differentiates a row of thick, densely aligned, mechanosensory bristles and a second row of thinner, curved, chemosensory bristles. The dorsal AWM produces one chemosensory bristle per five mechanosensory bristles. The ventral row is composed of thin bristles interspersed with chemosensory bristles in every fifth position. The alula is located in the posterior compartment. It produces a single row of long thin bristles along the margin on the ventral surface. The dorsal surface of the alula lacks bristles. The adult wing differentiates five longitudinal veins. L1 is located on both dorsal and ventral sides of the wing margin and L2-L5 veins are located in the wing blade. Veins L2-L5 are asymmetrical on the dorsal and ventral surfaces of the wing. One side contains more rows of tightly packed cells ('corrugated vein'). The opposite side is thinner ('ghost vein'). Corrugated veins consist of three rows of strongly pigmented and densely packed cells. Ghost veins consist of a single row of cells. Longitudinal veins L3, L5 and the distal tip of L4 are dorsally corrugated. Veins L2 and proximal L4 are ventrally corrugated (Milán, 2001b).
The msh gene belongs to the msh/Msx family of homeobox genes involved in dorsal cell fate specification in the Drosophila neuroectoderm. Since msh is expressed in the dorsal compartment of the wing disc, an investigation was carried out to see whether msh is also involved in dorsal identity specification in the Drosophila wing. For this purpose, msh mutant clones were generated in the wing and the DV identity of the bristles located along the AWM, in the alula and the DV corrugation of longitudinal veins in mutant cells, was assessed. Clones mutant for msh have no aberrant phenotype in the ventral surface of the wing. When mutant for msh, the dorsal anterior wing margin differentiates ventral bristles. A single row of thin bristles interspersed with chemosensory bristles in every fifth position is observed. Thus, the anterior wing margin differentiates a ventral pattern of bristles symmetrically on both surfaces (Milán, 2001b).
When covered with mutant cells, the dorsal surface of the alula differentiates bristles. This reflects transformation to a ventralized cell fate.
Absence of msh activity also induces a change in the pattern of corrugation of the longitudinal veins. In wild-type wings, veins L2 and L4 differentiate as 'ghost veins' on the dorsal surface. When mutant for msh, these veins are corrugated and differentiate three rows of strongly pigmented cells, thus mimicking a ventral-like pattern. Veins L3 and L5 are normally corrugated on the dorsal surface. When mutant for msh, they lose pigmentation and consist of a single row of aligned cells. Thus veins differentiate ventral characteristics in the dorsal surface when mutant for msh. It is concluded that msh is required in the dorsal compartment of the Drosophila wing to confer dorsal cell identity. In the absence of msh, symmetric wings are observed that differentiate ventral characteristics on both surfaces (Milán, 2001b).
Apterous is expressed in dorsal cells and is required to confer dorsal cell identity. It was therefore necessary to determine whether msh expression in the dorsal compartment is regulated by Apterous activity. MSH mRNA and msh-lacZ reporter genes are expressed in the dorsal compartment of the wing disc. MSH mRNA is expressed at a low level throughout the dorsal compartment, except in the region of the anterior margin where it is expressed at higher level. Ectopic expression of Apterous in the ventral compartment under control of dppGal4 induces ectopic expression of MSH mRNA at a level comparable to the overall low dorsal level. In apterous mutant discs, msh expression is lost from dorsal cells of the reduced wing pouch, but expression in the anterior mesopleura and hinge region remains. Finally, overexpression of dLMO, a repressor of Apterous activity in the Drosophila wing, represses expression of the msh-lacZ reporter gene. These results indicate that msh is indeed a target of Apterous. Additional studies show that ectopic expression of msh in the ventral surface is sufficient to confer dorsal identity on ventrally located cells (Milán, 2001b).
The results presented thus far indicate that mshis necessary and sufficient to specify dorsal identity in the Drosophila wing. A dominant mutation Dlw1 has been identified that shows partial dorsalization of the AWM. Both surfaces of Dlw1/+ AWMs have dorsal bristles, similar to what is observed when msh is ectopically expressed in the ventral compartment. Interestingly, Dlw alleles are associated with breakpoints located 30-90 kb upstream of the msh gene, raising the possibility that Dlw alleles may be regulatory mutants of msh. Indeed, a lethal allele of msh, mshDelta68, has proved to be lethal when heterozygous with Dlw1 and the recessive lethal alleles Dlw3 and
lw4. Dorsal clones mutant for Dlw3differentiate ventral structures. Genetic evidence is provided that supports the proposal that the msh gene is expressed in an Apterous-independent manner in Dlw1 wings (Milán, 2001b).
msh mRNA levels are reduced throughout the wing pouch in discs heterozygous for Dlw1. The low level of msh expression in the Dlw1 background may explain the loss of function characteristics exhibited by the Dlw1 allele in homozygous mutant clones. Dlw1/Dlw1 mutant clones located in the dorsal surface of the wing differentiate ventral structures. Thus, Dlw1 causes a dominant transformation of ventral cells to dorsal identity when heterozygous and an opposite transformation of dorsal cell to ventral identity when homozygous mutant in clones. Interestingly, the dominant mutation Drop, which affects eye development, has been recently shown to be a gain-of-function allele of msh (Mozer, 2001). Drop mutants contain lesions in the same region as Dlw mutants (i.e. upstream of the msh transcription start site) and ectopic expression of msh in the eye phenocopies the Drop phenotype. However, Mozer was not able to find detectable misexpression of msh in Drop mutants. Thus, undetectably low levels of msh misexpression in eye and wing seem to be associated with the dominant adult phenotypes associated with the Dlw and Drop alleles of msh (Milán, 2001).
Apterous activity is required to confer dorsal identity and dorsal-type signaling properties. Fringe and Serrate expression in dorsal cells induce a cascade of short-range interactions between dorsal and ventral compartments that lead to the expression of the organizing molecule Wingless along the DV compartment boundary. The results reported in this study suggest that msh confers dorsal identity without affecting DV signaling. In order to verify that this is the case, the ability of msh to restore dorsal identity and dorsal signaling properties in the absence of Apterous activity was examined. In apGal4/apUGO35 flies, the wing margin is reduced and the wing is considerably smaller than normal owing to reduced Apterous activity. When present, margin bristles have ventral identity in this genotype. Expression of msh in apGal4/apUGO35;uas-msh flies does not restore outgrowth of the wing. The few margin bristles observed in the dorsal surface of these wings have dorsal identity. Growth and wing margin formation can be restored in the apGal4/apUGO35 mutant background by expression of Fringe under apGal4 control. In these wings, both surfaces differentiate ventral structures: the AWM and the alula differentiate ventral bristles on both surfaces and the pattern of vein corrugation is ventral. Co-expression of msh with EP-fringe confers dorsal differentiation in the bristles of the dorsal AWM in these rescued wings. It was also noted that overexpression of msh in dorsal cells reduces the size of the dorsal wing pouch, induces differentiation of ectopic bristles in the wing blade and affects vein differentiation. This was also observed in apGal4/+; uas-msh/+ flies and presumably reflects defects caused by higher than normal Msh levels in dorsal cells. Note that the endogenous levels of msh expression in the wing pouch are quite low. These results suggest that developmental regulation of Msh protein levels may be crucial for proper wing development and differentiation of patterning elements. All these results indicate that msh confers dorsal identity without affecting dorsal signaling properties (Milán, 2001).
Two apterous homologs, Lmx1 and Lhx2, have been implicated in vertebrate limb development. Interestingly, these two genes appear to have separable functions in conferring dorsal identity and limb outgrowth. Lmx1 is expressed in the dorsal compartment of vertebrate limbs and is necessary and sufficient to confer dorsal identity. Lhx2 induces Radical-fringe expression in the apical ectodermal ridge, which is required for limb outgrowth. This contrasts with the situation in Drosophila where Apterous is responsible for both dorsal fate specification and for establishing the Fringe-dependent signaling center at the DV boundary. The findings reported here implicate msh as the principle target gene through which Apterous confers dorsal cell fate. msh is necessary and sufficient to induce dorsal cell fate, but has no role in DV boundary signaling. Intriguingly, the msh/Msx family of homeobox genes is also differentially expressed along the DV axis of the embryo and msh is required in the Drosophila neurectoderm to specify dorsal fate (Milán, 2001).
Individual neurons express only one or a few of the many identified neurotransmitters and neuropeptides, but the molecular mechanisms controlling their selection are poorly understood. In the Drosophila ventral nerve cord (VNC), the six Tv neurons express the neuropeptide gene FMRFamide (FMRFa). Each Tv neuron resides within a neuronal cell group specified by the LIM-homeodomain (LIM-HD) gene apterous (ap). The zinc-finger gene squeeze acts in Tv cells to promote their unique axon pathfinding to a peripheral target. There, the BMP ligand Glass bottom boat activates the Wishful thinking receptor, initiating a retrograde BMP signal in the Tv neuron. This signal acts together with apterous and squeeze to activate FMRFamide expression. Reconstituting this 'code,' by combined BMP activation and apterous/squeeze misexpression, triggers ectopic FMRFamide expression in peptidergic neurons. Thus, an intrinsic transcription factor code integrates with an extrinsic retrograde signal to select a specific neuropeptide identity within peptidergic cells (Allan, 2003).
FMRFa is specifically expressed in the six Tv neuroendocrine neurons located bilaterally in the three thoracic (T1-3) segments of the embryonic and larval VNC. apterous is expressed in three interneurons per VNC hemisegment, as well as in a lateral cluster of four neurons (the ap-cluster) in each of the T1-3 hemisegments. One of the four ap-cluster cells is the FMRFa-expressing Tv neuron. All ap interneurons in the VNC, except for the Tv, join a common ipsilateral axon tract termed the ap-fascicle. The Tv axon instead projects to the midline and exits the VNC dorsally to innervate the dorsal neurohemal organ (DNH). The DNH is a club-like neuroendocrine structure formed by two glial cells protruding from the midline of each thoracic segment. Anteriorly, two additional FMRFa-expressing cells are found, denoted SE2 cells. The SE2 cells do not express, nor depend upon, any regulators described in this study for their FMRFa expression. ap is important for the expression of FMRFa in the Tv neurons, but since most ap neurons do not express FMRFa, other regulators are likely needed for FMRFa regulation (Allan, 2003).
Rotund, a zinc finger protein of the C2H2 Krüppel-type belongs to a conserved subfamily of zinc finger proteins together with Drosophila CG5557, C. elegans Lin-29, and rat CIZ. Squeeze is most closely related to Rotund, with identity greater than 90% throughout the zinc finger region; Squeeze is 78% identical to LIN-29 in the conserved zinc finger region. Both rotund and CG5557 are expressed in subsets of cells in the developing CNS. CG5557 has a larval lethal phase. Mutants eclosed at a low frequency as immotile adults that died within 24 hr. Mutant larvae display a motility defect whereby the body wall musculature over-contract radially during the peristaltic wave typical of insect larval motility, apparent as a 'squeezing' of the intestine. Since this motility phenotype is fully penetrant and scored with 100% accuracy (sqzlacZ/sqzDf), CG5557 was renamed squeeze (sqz) (Allan, 2003).
The expression of sqz is largely restricted to subsets of cells in the CNS throughout embryonic and first instar larval (L1) development. Using sqzGAL4 to drive expression of the axonal reporter, UAS-τ-myc, sqz was found to be expressed in a population of lateral interneurons, primarily projecting axons in the anterior and posterior commissures. In sqz mutants, expressing neurons are generated and appear to extend axons along the appropriate tracts. Using both sqzlacZ and sqzGAL4, tests were performed for overlap with ap; sqz and ap were found to be co-expressed specifically within the thoracic ap cluster. Co-expression of sqz and ap is evident from the onset of ap expression at stage 14, with one neuron typically expressing higher levels of sqz. By stage 17, sqz expression is restricted to two neurons within the ap-cluster, with one neuron typically continuing to display higher levels of expression. Expression overlap between sqz and FMRFa was tested in late stage 17 embryos, when FMRFa expression commences; sqz is indeed selectively expressed at higher levels within the FMRFa Tv neuron. Thus, the six neurons within the VNC that co-express ap and higher levels of sqz selectively express the neuropeptide FMRFa and innervate the three specialized neuroendocrine glands -- the dorsal neurohemal organs (Allan, 2003).
To determine whether sqz regulates FMRFa expression, immunoreactivity for the FMRFa peptide was compared in wild-type and sqz mutant L1 larvae. In wild-type, FMRFa immunoreactivity is robust (98%) in all six Tv neurons. In sqz mutants (sqzlacZ/sqzDf), FMRFa staining was found to be reduced in all Tv neurons and was detected in 75% of cells. The T1 segment was most affected, with FMRFa expressed in 40% of T1 Tv neurons. To verify that the observed effects reflected regulation of the FMRFa gene, antibodies recognizing the C-terminal of the FMRFa precursor peptide (proFMRF) were used, as well as an FMRFa-lacZ reporter that faithfully reports FMRFa expression in Tv neurons. An equivalent effect on proFMRF (75%) and FMRFa-lacZ (77%) was found in sqz mutants (sqzlacZ/sqzDf and sqzGAL4/sqzDf, respectively) when compared to wild-type. Again, segment T1 is most affected with FMRFa-lacZ expressed in only 50% of T1 Tv neurons. These results show that sqz in part regulates the expression of the FMRFa gene in Tv cells (Allan, 2003).
To determine whether sqz regulates axon pathfinding of the Tv neuron, apGAL4 was used to drive the expression of a membrane-targeted reporter (UAS-EGFPF). In sqz mutants, a frequent failure of the Tv axon to innervate the DNH was observed, instead, it apparently joins the ap-fascicle. This phenotype is most pronounced within the most anterior thoracic segment (T1). In wild-type embryos, the DNH was innervated in 100% of thoracic segments, whereas sqz mutants (apGAL4/+;sqzie/sqzDf,UAS-EGFPF) show axonal innervation in 69% of T1 segment DNHs. Failure of innervation did not result from the absence of the DNH itself, since its profile was evident in affected segments. These results show that sqz is important for proper pathfinding of Tv axons and that the Tv axon often fails to diverge from the ap-fascicle in sqz mutants, apparently reverting to an “ap-only” phenotype (Allan, 2003).
Several determinants critical for proper FMRFa expression have been identified. These include a general peptidergic cell identity, co-expression of sqz and ap, axon projection out of the VNC, and competence to respond to a retrograde signal by activating the BMP pathway. When these criteria are met, either in the endogenous or ectopic case, FMRFa expression is triggered. Importantly, none of these events are individually exclusive to the Tv cell, but they are uniquely combined in only these 6 out of the 10,000 cells in the VNC. Reconstituting this scenario in other peptidergic neurons can trigger FMRFa expression. These results are in line with the emerging theme of a critical interplay between combinatorial transcription factor codes and signal transduction pathways in regulating gene expression and provide a clear example of how these general mechanisms also apply to the specific regulation of a terminal differentiation gene in the nervous system (Allan, 2003).
Why is ectopic FMRFa expression restricted to peptidergic neurons? Conceivably, cells responding to BMP activation and sqz/ap co-misexpression may arise from precursor cells utilizing a common genetic program, resulting in a chromatin state where the FMRFa gene is accessible to activation. Currently, the lineage from which most neuropeptidergic neurons arise is unknown, and any common theme behind their generation is uncertain. FMRFa expression may also be constrained by the presence of activators common to peptidergic neurons and/or by repressors present in non-peptidergic neurons. Common properties of peptidergic neurons, such as the dense core vesicle secretory machinery and the processing of precursor peptides, may indicate the existence of common regulatory programs for all peptidergic neurons. In support of this notion, recent studies of a novel basic helix-loop-helix transcription factor, dimmed, show that this gene is specifically expressed in most if not all peptidergic neurons. In dimmed mutants, peptidergic and secretory properties of the majority of peptidergic neurons are affected, including the expression of processing enzymes and several neuropeptides, such as FMRFa. This shows that dimmed plays a key role in specifying the peptidergic fate and supports the notion of a common regulatory program for this cell type (Allan, 2003).
Previous studies found that ap is essential for axon pathfinding of the majority of ap-neurons. However, ap does not affect Tv axon pathfinding, suggesting that the role of ap in Tv cells may exclusively be to regulate FMRFa expression. In line with these results, ap mutants do not show any apparent loss of pMad accumulation in the Tv neurons. In contrast, sqz mutants have Tv axon pathfinding phenotypes, and, consequently, a partial loss of pMad staining specifically in Tv neurons. Observations of Tv axons at the midline suggest that in the absence of sqz, the Tv axon likely reverts to an 'ap-only' axonal phenotype and turns to grow along the common ap-fascicle. Given the importance of DNH innervation for FMRFa expression, axon pathfinding defects in sqz mutants likely contribute to the loss of FMRFa in some hemisegments. However, the great difference in the loss of FMRFa expression between sqz (75%) and wit (0%) argues that sqz is not critical for BMP signaling, but rather affects it indirectly by affecting Tv axon pathfinding. Moreover, the sqz axon pathfinding phenotype is only partially penetrant and fails to explain either the reduction of FMRFa expression observed in all hemisegments, or the potency of sqz (acting together with ap) to trigger ectopic expression in Va and Vap peptidergic neurons (cells whose axons already exit the VNC and are pMad-positive). Misexpression of sqz in all ap cells occasionally leads to an additional pMad/FMRFa positive cell in the ap-cluster. In these cases, no ectopic FMRFa expression is detected in any axons extending in the common ap-fascicle, only in axons projecting into the DNH. Therefore, sqz misexpression likely alters the identity of another ap-cluster cell, imposing a Tv-like axonal pathfinding behavior and causing it to ectopically innervate the DNH. Thus, it appears that sqz regulates two critical features of Tv cell identity: differential pathfinding, and FMRFa expression (both directly and indirectly) (Allan, 2003).
Why do sqz and ap function to activate FMRFa expression within only three neuropeptidergic cell types (the Tv, Va, and Vap cells) which together comprise only 18 out of ∼200 peptidergic neurons in the developing Drosophila VNC? Using the specific GAL4 lines, apGAL4, VaGAL4, and VapGAL4 to drive the expression of UAS-EGFPF, it was found that all three neuronal subsets exit the VNC: Tv axons via the DNHs, Va axons via the transverse nerves, and Vap axons via the posterior A8 nerves. This observation is important in light of previous studies of tinman (tin) mutants. In tin mutants, a number of mesodermally derived tissues, including the DNHs, fail to develop. As a result, Tv axons stall at the presumptive midline exit point and, intriguingly, FMRFa expression is strongly reduced. This suggests that the DNHs may be necessary for proper FMRFa expression in Tv cells. These findings have been confirmed; in tin mutants, the DNHs are absent, and proFMRF staining is weak and only detected in 10% of Tv neurons. To address the putative target requirement for FMRFa expression in an alternative way, apGAL4 was used to express molecules that either alter Tv axon pathfinding or interfere with Tv axonal transport. roundabout (robo), a receptor that mediates repulsion from the VNC midline, was tested. In apGAL4/UAS-robo L1 larvae, Tv axons avoid the midline and fail to innervate the DNH. As predicted, this results in a loss (2%) of FMRFa-lacZ expression. Next, dominant-activated rac (UAS-racV12) was tested; it causes Tv axons to stall before reaching the midline and they fail to innervate the DNHs. This results in a complete loss (0%) of FMRFa-lacZ expression. To interfere with axonal transport, apGAL4 was used to express a dominant-negative version of the P150/Glued dynactin motor complex component (UAS-GluedDN), a molecule shown to specifically interfere with retrograde axonal transport. In apGAL4/UAS-GluedDN L1 larvae, a complete loss (0%) of FMRFa-lacZ expression was detected. Similarly, expression of the microtubule binding Tau protein, shown to interfere with axonal transport in Drosophila led to a near complete loss (4%) of FMRFa-lacZ expression (apGAL4/UAS-τ-myc). In both UAS-GluedDN and UAS-τ-myc, normal Tv axon innervation of the DNH was observed in all segments (Allan, 2003).
By co-expressing UAS-EGFPF in all scenarios outlined above, it was found that loss of FMRFa expression was not due to loss of the Tv cell, since the number of cells within the ap-cluster was unaltered in tin, UAS-robo, UAS-racV12, UAS-GluedDN, and UAS-τ-myc. Using α-Glutactin, it was found that the DNH itself is only affected in tin mutants, not in the other genotypes. Together, these results show that innervation of the DNH and retrograde signaling is essential for the expression of FMRFa (Allan, 2003).
What is the identity of the retrograde FMRFa-inducing signal? Recently, a Drosophila BMP type-II receptor, wishful thinking (wit), was implicated in mediating a retrograde signal from muscles to motor neurons, responsible for presynaptic maturation. Signaling by the TGF-β/BMP superfamily occurs via activation of a receptor complex, consisting of two type I and two type II receptors, leading to phosphorylation and nuclear translocation of a receptor Smad protein. In Drosophila, BMP signaling leads to the phosphorylation and nuclear translocation of the Smad protein Mothers against dpp (Mad), which can be monitored using antibodies specific to phosphorylated Mad (pMad) (Allan, 2003).
Using antibodies to pMad, BMP activation in peptidergic neurons was assayed. Nuclear pMad was detected not only in motor neurons, but also in the Tv, Va, and Vap neurons, demonstrating that peptidergic neurons projecting out of the VNC also show evidence of BMP activation. Accumulation of pMad in the Tv neurons commences during stage 17, immediately following DNH innervation. These results led to a test of whether Tv innervation of the DNH would be critical for pMad accumulation and consequently for FMRFa expression. Indeed, it was found that the absence of the DNH (in tin mutants), Tv axon pathfinding alterations (in apGAL4/UAS-robo and apGAL4/UAS-racV12) and interference with Tv axonal transport (in apGAL4/UAS-GluedDN and apGAL4/UAS-τ-myc) are all accompanied by loss of pMad staining specifically in Tv neurons. The ectopic ap-cluster FMRFa-expressing cell induced by sqz misexpression is also pMad positive. Given the role of sqz in Tv axon pathfinding, this is interpreted as resulting from sqz dominantly altering the projection of one other ap-cluster cell, forcing it to innervate the DNH. Thus, in all genotypes examined, Tv axonal projection to the DNH is critical for pMad accumulation (Allan, 2003).
Since Wit is expressed in a restricted pattern in the developing VNC, attempts were made to address whether the Tv neurons express Wit. However, single-cell resolution could not be obtained with the Wit antibody and Wit could not be definitely localized in Tv cells. However, the wit-dependent pMad accumulation in Tv neurons, the apGAL4/UAS-tkvA, UAS-saxA-mediated rescue of wit mutants, and the UAS-gbb-mediated 'rescue' of UAS-robo misexpression, provide genetic evidence supporting the expression of wit in Tv cells. Previous studies have shown that gbb is expressed in developing endoderm and visceral mesoderm, but it has not been detected in the VNC. By in situ hybridization, no apparent expression was detected in the DNH. Given that the DNH only contains two cell bodies, low-level gbb expression may be beyond detection. Moreover, since the anterior midgut is positioned in very close proximity to the DNHs, it is possible that Gbb diffuses from the visceral mesoderm to the DNH (Allan, 2003).
Why is BMP activation necessary for FMRFa expression? Neither forced axonal exit from the VNC (apGAL4/UAS-Unc5) nor autocrine presentation of the Gbb ligand (apGAL4/UAS-gbb) leads to activated pMad and FMRFa expression in ap cells other than the Tv cell. This indicates that the Tv cell is uniquely predetermined to respond to the Gbb ligand. In fact, even direct activation of the BMP pathway (UAS-saxA, -tkvA;apGAL4/+) in all ap neurons does not trigger ectopic FMRFa expression, showing that the Tv cell is further uniquely capable of responding to BMP activation. The misexpression results show that both of these properties of the Tv cell are specified by sqz/ap co-expression. Given this level of Tv cell predetermination, it begs the question as to why Tv cell FMRFa expression evolved to be dependent upon a retrograde BMP signal. Perhaps dependence upon a retrograde signal provides precise control over the onset of FMRFa expression during embryogenesis. In fact, Tv neurons are born by stage 14 (as evident by ap expression) but do not activate FMRFa expression until late stage 17, upon DNH innervation. Alternatively, the presence of a small number of sqz/ap co-expressing cells in the developing brain that do not express FMRFa may necessitate additional regulatory control over FMRFa expression. Dependence upon a signal transduction pathway also provides several unique means of control and amplification of target gene expression. Finally, the fact that sqz, ap, and BMP activation only act to trigger FMRFa expression within a neuropeptidergic cellular context reveals additional complexity underlying the control of specific neuropeptide expression. Given the large number of diverse cell types in the CNS, what may appear to be an almost excessive complexity of combinatorial coding may in fact be essential for high fidelity of gene expression (Allan, 2003).
A set of peptidergic neurons is conserved throughout all developmental stages in the Drosophila central nervous system. A small complement of 28 apterous-expressing cells (Ap-let neurons) in the ventral nerve cord (VNC) of Drosophila larvae co-express numerous gene products. The products include the neuroendocrine-specific bHLH regulator called Dimmed (Dimm), four neuropeptide biosynthetic enzymes (PC2, Fur1, PAL2, and PHM), and a specific dopamine receptor subtype (dDA1). For the PC2, Fur1, and PAL2 enzymes, and for the dDA1 receptor, this neuronal pattern represents the vast majority of their total expression in the VNC. In addition, while Dimm and PHM are present in the peritracheal Inka cells in larvae, pupae, and adults, Ap, PC2, Fur1, PAL2, and dDA1 are not. PC2, PAL2, and DA1 receptor expression are all controlled by both dimm and ap. Previous genetic analysis of animals deficient in PC2 revealed an abnormal larval ecdysis phenotype. Together, these data support the hypothesis that the small cohort of Ap-let interneurons regulates larval ecdysis behavior by secretion of an unidentified amidated peptide(s). This hypothesis further predicts that the production of the Ap-let neuropeptide(s) is dependent on each of four specific enzymes, and that a certain aspect(s) of its production and/or release is regulated by dopamine input (Park, 2004).
In the Drosophila ventral nerve cord, a small number of neurons
express the LIM-homeodomain gene apterous (ap). These ap
neurons can be subdivided based upon axon pathfinding and their expression of
neuropeptidergic markers. ap, the zinc finger gene squeeze, the
bHLH gene dimmed, and the BMP pathway are all required for proper
specification of these cells. Here, using several ap neuron terminal
differentiation markers, how each of these factors contributes to ap neuron diversity has been resolved. These factors interact genetically and biochemically in subtype-specific combinatorial codes to determine certain defining aspects of ap neuron subtype identity. However, it was also found that ap, dimmed, and squeeze additionally act independently of one another to specify certain other defining aspects of ap neuron subtype identity. Therefore, within single neurons, single regulators acting in numerous molecular contexts differentially specify multiple subtype-specific traits (Allan, 2005).
Within every VNC hemisegment, ap is expressed by one dorsal neuron (dAp) and two ventral neurons (vAp). Additionally, in thoracic VNC hemisegments, ap is expressed by a lateral cluster of four neurons (the ap cluster), termed the Tv, Tvb, Tva, and Tvc neurons. These ap neurons are phenotypically
diverse. The axons of most ap neurons project within an ipsilateral
fascicle (ap fascicle) that projects to the brain, whereas the axons of the Tv cell exit the VNC at the midline to innervate the dorsal neurohemal organs (DNH).
A subset of ap neurons is peptidergic (the Tv, Tvb, and dAp
neurons). As is characteristic for the vast majority of Drosophila peptidergic
neurons, these cells express high levels of the peptide biosynthetic enzyme
peptidylglycine alpha-hydroxylating monooxygenase (PHM).
However, this peptidergic subset is also diverse: Tv cells selectively express
the dFMRFa neuropeptide, whereas Tvb and dAp cells selectively coexpress three
peptide biosynthetic enzymes -- PC2, Furin1, and PAL2 -- although the
identity of their secreted neuropeptide(s) remains unknown. This coexpression in
Tvb and dAp cells suggested a functional grouping and a common name, 'Ap-let'
cells. For clarity, the ap neurons will be considered as three
classes: (1) Tv cells express dFMRFa and PHM and innervate the DNH; (2) Ap-let (Tvb and dAp) cells express PHM, PC2, Furin1, and PAL2; (3) the vAp, Tva, and Tvc cells are nonpeptidergic (Allan, 2005).
ap, sqz, dimm, and the BMP pathway act in a
combinatorial code to regulate dFMRFa in the Tv cell (ap,
sqz, dimm, and the BMP pathway) and furin1 (ap,
dimm) in Ap-let cells. Importantly, however, each
regulator also plays critical roles within these ap neurons independent
of the other regulators. Ap independently acts to regulate axon pathfinding by
all ap cells except the Tv. Dimm independently controls PHM in the Tv and
Ap-let cells. Moreover, Sqz independently acts via the N pathway to regulate
cell identity within the ap cluster, upstream of both Ap and Dimm,
apparently by suppressing the Tvb cell fate to favor the Tv fate. The Ap-let
cells do not express Sqz, nor do they have an activated BMP pathway. In these
neurons, Ap activates the expression of Dimm, and both act together to activate
the expression of the peptide-processing enzyme Fur1. The Tva and Tvc cells of
the ap cluster do not express Dimm and do not have an activated BMP
pathway. Remarkably, the differences inferred between regulatory circuits for the
two classes of peptidergic cells are highly reminiscent of differences in
regulatory circuits that operate during the differentiation of distinct
noradrenergic neurons. Together, these sets of studies support the proposition
that epistatic relations between regulators underlying the production of a
common phenotype may differ according to cell type (Allan, 2005).
The loss-of-function and gain-of-function phenotypes presented for ap, sqz, dimm, and the BMP pathway,
suggest that they act in a combinatorial fashion to regulate dFMRFa
expression in the Tv neuron. Likewise, the results indicate that ap and
dimm, in the absence of sqz and the BMP pathway, combine to
activate Fur1 in the Ap-let neurons, Tvb and dAp. In order to determine whether
these regulators act simultaneously on dFMRFa and Fur1, rather than in a genetic
hierarchy, the epistatic and biochemical relationship between
these regulators were studied. Only one clear epistatic relationship was found;
Ap activates the expression of Dimm in the majority of ap neurons. Therefore, it was important to determine whether Dimm acted downstream of Ap to independently and more directly regulate dFMRFa and Fur1 expression. This hypothesis was tested in two complementary tests. (1) Rescuing Dimm function in ap neurons that were absent for Ap function, yielded a nearly complete rescue of dFMRFa in Tv neurons, but only relatively weak rescue of Fur1 in Ap-let neurons.
(2) Panneuronal co-misexpression of both ap and dimm
triggers ectopic dFMRFa expression in a much greater number of neurons than does
either regulator alone. These data indicate that Dimm functions together with Ap
to achieve wild-type levels of dFMRFa and, more notably, Fur1. Thus, ap
and dimm appear to display both hierarchical and combinatorial
interactions. This hypothesis has precedent in studies of the developing
pancreas, in which Foxa2 is required for pdx-1 transcription in β
cells and later interacts directly with PDX-1 protein to regulate target gene
expression, including maintained pdx-1 expression. Biochemical data are
also consistent with the possibility that a combinatorial Ap, Dimm, and Sqz code
that activates dFMRFa and dFur1 involves direct protein
interactions. These may exist within larger complexes bridged by Chip, since Dimm
can interact directly with both Ap and Chip, and this in turn may explain why
Dimm partially rescues both the ap mutant dFMRFa and Fur1 phenotypes.
These multiple interactions are reminiscent of
synergistic interactions suggested between mammalian bHLH proteins, LIM-HD
proteins, and the Chip homolog, LDB1/NLI. The simplest explanation for restricted
patterns of neuropeptides and certain neuropeptide biosynthetic enzymes features
a combinatorial hypothesis. More specifically, it is proposed that different
combinatorial codes of transcription factors act cell specifically to effect
differing patterns of neuropeptides and associated processing enzymes (Allan, 2005).
Ap expression is an early marker of ap cell
differentiation, and it is required for proper axonal pathfinding by most
ap neurons, although not by the Tv cell. In contrast, neither Sqz nor
Dimm appear to control ap cell morphogenesis. An independent role for Sqz
occurs early in ap cell differentiation, at a time when postmitotic
cell fates are being determined. It is surprising that such cell fate changes
can be rescued by UAS-Dl. Why would the frequently used N pathway
signaling system depend upon a much more restricted regulator like sqz
for proper activity? Increasing evidence points to major mechanistic differences
between N signaling during neuroblast specification and during asymmetric
division, where asymmetric divisions specifically require neuralized,
numb, and sanpodo. No expression of sqz is found in
neuroblasts, but expression is evident in many VNC cells. Therefore, it is proposed
that factors like Sqz coordinate late N signaling with cell specification and/or
cell cycle genes (Allan, 2005).
Dimm acts independently of Ap, Sqz, and
the BMP pathway to activate expression of the neuropeptide-processing enzyme
PHM. The evidence regarding the independent role of Dimm suggests that it is a
master regulator of neuroendocrine cell fate. dimm expression is highly
correlated with a neuroendocrine/peptidergic cellular identity, where it
regulates the expression of almost all neuropeptides and their processing
enzymes examined to date, especially within those neurons that express peptides
that are processed to include an α-amidated C terminus. This is a
significant cellular pattern, because more than 90% of Drosophila
neuropeptides are amidated. Furthermore, high-level expression of the PHM enzyme is
absolutely required for amidation and serves as an excellent marker for most peptidergic neurons
in Drosophila. Finally, PHM expression appears to be dedicated to
neuroendocrine peptide biosynthesis; it is exclusively found within the
luminal domain of secretory vesicles. Thus, PHM expression provides a faithful
marker for the peptidergic/neuroendocrine cell fate. This study has shown that
PHM is dominantly induced by dimm overexpression throughout most or all
of the CNS. This evidence, together with the loss-of-function data argues strongly that
dimm is a neuroendocrine master regulator, with properties akin to those
of other bHLH proteins in regulating cell fate (Allan, 2005).
As anticipated,
more restricted peptidergic traits such as dFMRFa and Fur1 expression are
dependent upon combinatorial codes. Importantly, however, the
selection of cell-specific peptidergic markers arises from a deterministic
interaction between a peptidergic master regulator and a cell-specific
combinatorial code. There exists a clear analogy between the action of
dimm in developing neurons and results regarding the glial cells
missing (gcm) gene. Studies have shown that gcm is both
necessary and sufficient for glial cell specification within the
DrosophilaVNC.
gcm is able to ectopically activate generic glial genes, such as
reversed polarity, and also activates subclass-specific glial genes,
but only in certain prescribed subsets of cells. Thus, similar to gcm,
it is predicted
that dimm is a master regulator of core neuroendocrine genes in most
peptidergic/neuroendocrine cells. It will be of interest to determine which
genes beyond PHM are under dimm control. In parallel, dimm
combines with local-acting factors to help activate subclass-specific genes
(e.g., neuropeptide-encoding genes) within peptidergic cell subsets (Allan, 2005).
The genes studied here
combine to regulate dFMRFa and Fur1 but also have independent roles
within the same cells. This raises the issue of how Dimm, for instance, can
complex with Ap/Sqz on dFMRFa and also act independently on PHM
within the same nucleus. Surprisingly, no clear evidence of an
antagonistic relationship between the individual roles of Ap, Sqz, and Dimm was found. For
example, co-misexpression of ap with dimm does not obviously
suppress the ectopic PHM expression observed when dimm alone is
misexpressed. Likewise, misexpression of sqz in the Fur1-expressing
dAp/Tvb cells does not suppress Fur1. Thus,
it appears that the independent mechanisms of regulator action are robust and
can coexist with combinatorial functions. Therefore, it is proposed that these
regulators operate within a bistable organizational mechanism. With respect to
independent roles, it is proposed that Dimm operates independently of Ap and Sqz to
dominantly induce specific target genes (e.g., PHM) within all neuronal
lineages by forming heterodimers with a class A bHLH like Da, or by forming
homodimers. The Drosophila bHLH Twist
protein has distinct regulatory roles in vivo, acting either as a heterodimer
with Da, or as a homodimer. Notably, the mammalian ortholog of Dimm, Mist1, forms functional
homodimers to promote the differentiation of pancreatic secretory cells (Allan, 2005).
The TGFβ/BMP signal transduction pathway plays critical
roles during a number of developmental events, and mutants affecting the
Drosophila BMP pathway show dramatic defects in embryonic development. In
contrast, in the Tv neuron, BMP signaling plays a much more subtle
role, and although it is critical for dFMRFa expression, no effects were found
upon the expression of sqz, ap, or dimm or on the general
peptidergic marker PHM in wit mutants. Although these studies cannot rule
out other roles for the BMP pathway in Tv neurons, it is tempting to speculate
that target-derived BMP signaling in neurons may have quite a limited set of
nuclear readouts in each specific neuronal subclass (Allan, 2005).
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).
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).
One of the most widely studied phenomena in the establishment of neuronal identity is the determination of neurosecretory phenotype, in which cell-type-specific combinatorial codes direct distinct neurotransmitter or neuropeptide selection. However, neuronal types from divergent lineages may adopt the same neurosecretory phenotype, and it is unclear whether different classes of neurons use different or similar components to regulate shared features of neuronal identity. This question was addressed by analyzing how differentiation of the Drosophila larval leucokinergic system, which is comprised of only four types of neurons, is regulated by factors known to affect expression of the FMRFamide neuropeptide. All leucokinergic cells express the transcription factor Squeeze (Sqz). However, based on the effect on LK expression of loss- and gain-of-function mutations, three types of Lk regulation are described. In the brain LHLK (lateral horn leukokinin) cells, both Sqz and Apterous (Ap) are required for LK expression, but surprisingly, high levels of either Sqz or Ap alone are sufficient to restore LK expression in these neurons. In the suboesophageal SELK cells, Sqz, but not Ap, is required for LK expression. In the abdominal ABLK neurons, inhibition of retrograde axonal transport reduces LK expression, and although sqz is dispensable for LK expression in these cells, it can induce ectopic leucokinergic ABLK-like cells when over-expressed. Thus, Sqz appears to be a regulatory factor for neuropeptidergic identity common to all leucokinergic cells, whose function in different cell types is regulated by cell-specific factors (Herrero, 2007).
It has been shown that Ap is required for LK expression only in LHLK cells. Ap is also necessary for proper transcription of the Fmrf gene in the thoracic Tv neurons. In attempts to understand the mechanisms underlying leucokinergic differentiation, it was asked whether other factors known to control expression of the FMRFamide neuropeptide, i.e., Sqz and the BMP signalling pathway, affected LK expression. Indeed, the number of LK-immunopositive cells is strongly reduced in sqzlacZ mutant larvae. It has been proposed that Apterous is not necessary for the emergence and maintenance of LHLK cells. Earlier reports have established that the Tv cells are present in sqz mutants, although they do not express Fmrf. Are the LK cells present in sqz mutants? This question could not be directly addressed due to the lack of independent markers for following the fate of the LK cells, but the results presented in this study indicate that in sqz mut