Transcriptional Regulation Table of contents

Wingless expression in the leg and eye discs

The defects in legs and wings appear to arise through dose-dependent effects of dpp on wingless expression. A high level of dpp in the wing disc causes a reduction of wg expression in the presumptive scutellar region. Intermediate dpp levels in leg discs induces the expansion of wg expression into the ventral outgrowth, while high dpp expression eliminates ventral wg expression. It is thought that a critical role for dpp in leg and wing discs is to reduce or eliminate the expression of wg. Consistent with this role of dpp is the observation that ectopic wg expression is detected in imaginal discs when dpp signaling is impaired by lowering the activity of thick veins, one of the three known dpp receptors. Reciprocal interactions between wg and dpp appear to be important in the subdivision of the leg disc between dorsal and ventral compartments. Inhibition of wg expression by dpp in the dorsal anterior portion of the leg disc would help limit the intersection of dpp and wg expression to one site, providing a single distal organizer for the disc (Morimura, 1996).

wingless is transcribed in a ventral sector of the leg disc throughout development while dpp is transcribed in a stripe that spans the disc, although its expression is more intense dorsally than ventrally. DPP and Wingless specify respectively dorsal and ventral cell fates in the leg disc. It is suggested that expression of the ventral DPP half-stripe is nonfunctional and that inhibition of ventral DPP function could be mediated by WG. It is proposed that DPP specificies cell position relative to the dorsal midline in the leg disc, a role analogous to the role of DPP in the early embryo where it specifies fates within the dorsal 40% of the ectoderm relative to the dorsal midline (Held, 1994).

The dorsoventral midline of the Drosophila eye disc is a source of signals that stimulate growth of the eye disc, define the point at which differentiation initiates, and direct ommatidial rotation in opposite directions in the two halves of the eye disc. This boundary region seems to be established by the genes of the iroquois complex, which are expressed in the dorsal half of the disc and inhibit fringe expression there. Fringe controls the activation of Notch and the expression of its ligands, with the result that Notch is activated only at the fringe expression boundary at the midline. The secreted protein Wingless activates the dorsal expression of the iroquois genes. Pannier, which encodes a GATA family transcription factor expressed at the dorsal margin of the eye disc from embryonic stages on, acts upstream of wingless to control mirror and fringe expression and establish the dorsoventral boundary. Loss of pannier function leads to the formation of an ectopic eye field and the reorganization of ommatidial polarity, and ubiquitous pannier expression can abolish the eye field. Pannier is thus the most upstream element yet described in dorsoventral patterning of the eye disc (Maurel-Zaffran, 2000).

The pnr gene is expressed in the dorsalmost embryonic cells, in a domain of the notum surrounding the dorsal midline, and at the dorsal anterior margin of the eye disc. The FLP-FRT system was used to generate clones of cells mutant for pnrVX6 , a null allele. Mutant clones were produced in the eye disc using the yeast FLP recombinase expressed under the control of the eye-specific enhancer of eyeless. Only clones produced at the dorsal margin of the eye disc give rise to a phenotype. In such discs an ectopic field of differentiating photoreceptors appears anterior to the main eye field. In adult flies this results in the formation of an ectopic eye field in the dorsal head cuticle, which is either separate from or fused with the normal eye. Interestingly, these ectopic eye fields do not arise exclusively from the pnr mutant cells within the clone itself, but also contained a domain of wild-type cells. These observations suggested that the new boundary of pnr expression present at the edge of the clone could be responsible for the induction of this new eye field. Frequently a duplication of the antenna is also observed, probably reflecting the function of pnr expressed dorsally in the antennal disc (Maurel-Zaffran, 2000).

To test the hypothesis that the boundary of pnr expression, rather than the absence of pnr, could be important for promoting eye growth, all pnr function in the eye was removed. Adult eyes and eye discs were examined from flies containing very large pnr mutant clones. In some cases, a dramatic loss of the eye and an absence of differentiating photoreceptors in the eye disc, resulting from the loss of all pnr function, were observed. In other cases eye overgrowth was observed; probably these eye discs retain some pnr-expressing cells, allowing the establishment of an ectopic pnr expression boundary. Only a small percentage of adults with large pnr clones were recovered; most animals died as late pupae and their heads were sometimes entirely missing, probably due to loss of all tissues deriving from the eye-antennal disc. A similar phenotype has been reported for some hypomorphic combinations of pnr alleles (Maurel-Zaffran, 2000 and references therein).

In the notum, the activity of pnr as a transcriptional activator is inhibited by binding to the zinc finger protein U-shaped (Ush), which is expressed in an adjacent domain. Ush does not appear to be required in the eye disc, since clones mutant for ush develop normally even when they are very large. However, ectopically expressed ush is able to inhibit the function of pnr in the eye disc; expression of ush with a pnr-GAL4 driver results in phenotypes similar to those induced by pnr mutant clones. Thus pnr is likely to act by activating the transcription of target genes in the eye as well as in the notum (Maurel-Zaffran, 2000).

The pnr boundary was eliminated by inducing ubiquitous pnr expression using the UAS/GAL4 system. A form of pnr was used that is resistant to inhibition by Ush, but appears to behave like wild-type pnr in the absence of Ush function. As a consequence, a complete loss of the eye is observed, confirming the importance of the border of pnr expression. From these experiments, it is concluded that the dorsally restricted expression of pnr is critical for eye development. A boundary between pnr-expressing cells and pnr-non-expressing cells appears to be necessary to induce growth and differentiation of the eye field (Maurel-Zaffran, 2000).

Recently, several studies have established that N activation along the dorsoventral midline of the eye disc is critical for eye growth as well as for positioning the equator. This local activation is the consequence of the ventrally restricted expression of fng, which is negatively controlled by the iro-C homeobox genes expressed in the dorsal half of the eye disc. Either loss of fng function, or ubiquitous expression of fng, caup or mirr, abolishes eye growth. The iro-C genes appear to act redundantly, as both ara and caup must be removed from clones of cells to promote the formation of ectopic dorsal eyes similar to those reported for pnr. The similar effects observed for gain or loss of pnr function suggest strongly that pnr might act in the same pathway as the iro-C and fng. To confirm this and to order pnr with respect to these genes, expression of mirr and fng was examined in eye discs mutant for pnr or misexpressing pnr. In eye discs in which pnr function had been removed, mirr expression is greatly reduced, whereas fng is derepressed dorsally. In eye discs expressing constitutively active pnr, mirr expression is expanded ventrally, shifting the point of morphogenetic furrow initiation to the ventral side. fng expression is dramatically reduced in discs overexpressing pnr D4. It thus appears that pnr acts upstream of the iro-C genes, activating their expression dorsally. Consistent with this, it has been found that ubiquitous expression of ara abolishes photoreceptor differentiation, and that removal of pnr function does not restore photoreceptor formation. If pnr were downstream of ara, blocking its function should have induced ectopic eye development even in the presence of ara (Maurel-Zaffran, 2000).

The results above show that pnr acts upstream of the iro-C genes to regulate dorsal eye development. Another molecule that has been shown to act upstream of the iro-C in this context is Wg. wg is required to inhibit the initiation of the morphogenetic furrow at the lateral margins of the eye disc, preventing ectopic eye differentiation there. The dorsal ectopic eyes induced by removing pnr function thus suggest that the functions of pnr and wg may be related. Consistent with this idea, the block in morphogenetic furrow initiation caused by expressing wg throughout the eye disc, like the block caused by expressing pnr D4 , can be rescued by co-expressing an activated form of N. pnr and wg may thus act in the same cascade to prevent eye differentiation (Maurel-Zaffran, 2000).

In situ hybridization has been used to show that pnr mRNA is restricted to the dorsal margin of the eye disc, anterior to and overlapping the morphogenetic furrow. wg is expressed at the dorsal and ventral edges of the eye disc with stronger expression dorsally, and its dorsal domain of expression resembles that of pnr. To test the epistatic relationship between wg and pnr, PNR mRNA expression was examined in eye discs from which wg function had been removed. Adult flies carrying wg minus clones show a transformation of the dorsal head cuticle into ectopic eye tissue, as well as missing antennae. Eye-antennal discs carrying such clones were identified by a severe reduction in the size of the antennal disc. In these eye discs PNR mRNA expression is wild type, showing that wg is not required for pnr expression. Overexpression of either Wg or an activated form of Armadillo (Arm), a downstream component of the Wg pathway, has no effect on pnr expression. Thus, wg is neither necessary nor sufficient for pnr expression. When pnr mutant clones are produced, dorsal wg expression is lost. Conversely, when a form of Pnr that is resistant to inhibition by U-shaped is overexpressed, although the size of the eye disc is dramatically reduced, and a derepression of wg expression is observed in both the eye and the antennal discs. It is concluded that pnr indeed activates wg expression at the dorsal margin (Maurel-Zaffran, 2000).

The role of wg in directing dorsal development is unexpected because wg is also expressed at the ventral anterior margin of the eye disc, although at a lower level than at the dorsal margin; this expression must have an upstream regulator other than pnr. However, the effects of loss of wg are more robust on the dorsal than the ventral side of the eye disc, and misexpression of wg symmetrically at both lateral margins dorsalizes the eye disc. These observations may be explained by the finding that at early stages wg is limited to the dorsal side of the eye disc and may exert its dorsalizing effect at this time (Maurel-Zaffran, 2000).

In Drosophila the eye-antennal disc gives rise to most adult structures of the fly's head. Yet the molecular basis for its regionalization during development is poorly understood. homothorax is shown to be required early during development for normal eye development and is necessary for the formation of the ventral head capsule. In the ventral region of the disc are homothorax and wingless involved in a positive feedback loop necessary to restrict eye formation. homothorax is able to prevent the initiation and progression of the morphogenetic furrow without inducing wingless, which points to homothorax as a key negative regulator of eye development. In addition, the iroquois-complex genes are shown to be required for dorsal head development, antagonizing the function of homothorax in this region of the disc (Pichaud, 2000).

The eye-antennal disc is a compound imaginal disc that gives rise to several different parts of the fly head: the head capsule (ventral and dorsal, including the ocellar region), that surrounds the eye, plus the antenna and maxillary palp. Because the gene homothorax (hth) has been implicated in limiting the eye field, its pattern of expression throughout the development of the eye-antennal disc was examined and its requirements in the head were examined in detail. Early in development, during the second instar larval stage, hth is weakly expressed in all the cells of the eye-antennal disc. The same widespread expression pattern is seen for wg. During the third instar larval stage, as the eye field is patterned in a posterior-to-anterior direction, the expression of hth regresses anteriorly and laterally. By late third instar, hth remains strongly expressed in the prospective head capsule, antennal regions and more weakly in the maxillary palp primordium. In the developing eye field, hth is expressed 10-15 cell diameters ahead of the morphogenetic furrow (MF), but it is switched off thereafter. In the differentiated eye, hth is found in the pigment cells in the posterior region of the eye field (Pichaud, 2000).

The progression of the MF orchestrates a wave of cell differentiation that gives rise to the different cell subtypes found in the adult eye. The product of the gene wingless (wg) limits the expansion of the differentiating eye, allowing head capsule development. Since hth also limits eye development in the ventral head, a test was performed to see if hth and wg regulation is linked. In third instar eye-head region, hth is strongly expressed in the prospective regions of the dorsal and ventral head, where it overlaps with wg expression. Large ventral hth-M1 mutant clones induced in the prospective head capsule region cause the formation of ectopic eyes, visualized by the de novo expression of Elav, and the loss of wg expression. Ectopic eye differentiation starts at the margins and progresses inward, in agreement with the ectopic eye tissue seen in the adult. By contrast, large dorsal hth clones do not produce ectopic eyes and wg expression is not affected. Nonetheless, removal of hth from the dorsal head results in the loss of the ocellar region. It has been shown that this region requires the expression of wg and its downstream target orthodenticle (otd). Thus, it is possible that, in the absence of hth, wg expression is subtly reduced. Alternatively, hth could act in parallel to, or downstream of wg to specify this dorsal head region. These results suggest that, in the ventral part of the eye- head region, hth helps defining the territory of the disc that will become head, probably in part by maintaining wg, but does not do so in the dorsal region. In the absence of hth, wg is lost ventrally and ectopic eyes are generated (Pichaud, 2000).

As the eye field is patterned, hth expression is repressed several rows of cells ahead of the MF, and it is upregulated at the margins of the disc. This dynamic pattern of expression resembles wg expression, which is detected in all the cells of the eye-antennal disc during second instar larval, but is later restricted to the margins. During late third instar, hth expression straddles that of wg. This observation raises the possibility that hth could be also controlled by wg. To test this hypothesis, clones were generated ectopically expressing a membrane-tethered Wg form (teth-Wg), that cannot diffuse. In teth-Wg expressing clones located anterior of the furrow, Hth levels are increased. This is not the case when the clones are induced in regions immediately in front of or posterior to the furrow. A test was performed to see if blocking the wg pathway could lead to modifications of hth expression. This issue was addressed by producing ectopic clones of a dominant negative form of dTCF (Pangolin), a nuclear factor required for the transduction of the wg signal. In these clones hth expression is strongly reduced in the presumptive head cuticle region both ventrally and dorsally. This result shows that wg is necessary to maintain hth expression in the presumptive head regions of the eye-head disc. Conversely, when hth is expressed ectopically in clones, it is unable to initiate wg expression. However, ectopic hth upregulates wg in regions where it (and hth) are already expressed. Also, ectopic expression of hth can block furrow initiation without inducing wg. These observations raise the possibility that hth mediates, at least partially, the eye-repressing function of wg (Pichaud, 2000).

These experiments suggest that hth is necessary to maintain wg expression, but not sufficient for its de novo induction. Starting during late second or early third instar larval stage wg and hth seem to be engaged in a positive regulatory feedback loop that might be important for the development of the ventral head capsule. This feedback loop could be responsible for the upregulation of hth in the ventral and dorsal head capsule while hth is required to maintain wg only in its ventral part (Pichaud, 2000).

Loss of DPP signaling in the Drosophila eye can lead to ectopic expression of wingless, suggesting that MAD negatively regulates wingless transcription. Mutant Mad clones are found to express wingless in eye imaginal discs. Similarly, bifurcations of antennae are associated with mutant Mad clone. Such clones express wg and are overgrown when located in the dorsal region of the antennal disc. Thus the antagonistic effect of DPP signaling in wg expression is also observed in other discs and might be a general mechanism during Drosophila imaginal disc development (Wiersdorff, 1996).

Signaling by the secreted Hedgehog, Decapentaplegic and Wingless proteins organizes the pattern of photoreceptor differentiation within the Drosophila eye imaginal disc; hedgehog and decapentaplegic are required for differentiation to initiate at the posterior margin and progress across the disc, while wingless prevents it from initiating at the lateral margins. Wingless and Decapentapegic often have opposing functions in differentiation. This is true in the eye imaginal disc, where Dpp effects are positive, promoting initiation of the morphogenetic furrow and also promoting photoreceptor differentiation. Wg effects are negative, opposing the effects of Dpp, inhibiting furrow initiation and likewise inhibiting differentiation. An analysis of these interactions has shown that initiation requires both the presence of decapentaplegic and the absence of wingless, which inhibits photoreceptor differentiation downstream of the reception of the decapentaplegic signal. The eyes absent and eyegone genes encode members of a group of nuclear proteins required to specify the fate of the eye imaginal disc. Both eyes absent and eyegone are required for normal activation of decapentaplegic expression at the posterior and lateral margins of the disc and also repression of wingless expression in presumptive retinal tissue. The requirement for eyegone can be alleviated by inhibition of the wingless signaling pathway, suggesting that eyegone promotes eye development primarily by repressing wingless. These results provide a link between the early specification and later differentiation of the eye disc (Hazelett, 1998).

As a direct test of the requirement for dpp in furrow initiation and of the inhibitory role of wg, clones of cells were generated of doubly mutant for Mothers against dpp (required to transduce the dpp signal) and wg were examined. Cells in these clones are unable to respond to dpp, but are also unable to produce wg. When such clones of cells occur at the posterior margin of the eye disc, they autonomously fail to initiate photoreceptor development. Clones of cells singly mutant for Mad also fail to differentiate as photoreceptors, but often have an additional non-autonomous inhibitory effect on photoreceptor differentiation by surrounding cells. This inhibitory effect is likely to be mediated by wg. Thus dpp signaling is required not only to repress wg expression, but also independently for morphogenetic furrow initiation (Hazelett, 1998).

wg is shown to inhibit photoreceptor formation downstream of the dpp receptors. wg is known to be required to prevent ectopic morphogenetic furrow initiation from the lateral margins of the eye disc. However, the mechanism by which wg inhibits photoreceptor differentiation is not well understood. It has been suggested that wg acts by preventing dpp expression, since dpp expression is lost in clones of cells lacking the kinase encoded by shaggy/zeste-white 3 (sgg), which normally functions to inhibit the wg pathway. However, a low level of ectopic wg can inhibit photoreceptor differentiation without reducing dpp expression. Since dpp positively autoregulates its own expression, inhibition of dpp function may result in a loss of dpp expression. If wg acted by inhibiting dpp expression, it should be possible to overcome its effects by expressing dpp from a heterologous promoter. However, co-expression of dpp and wg does not allow initiation of photoreceptor development at the posterior margin. Ectopic expression of dpp in the eye disc has been shown to specifically induce initiation of photoreceptor differentiation from the anterior margin of the disc in a non-autonomous fashion. Surprisingly, this ectopic differentiation is not inhibited by wg signaling. Co-expression of dpp and wg throughout the disc results in initiation from the anterior margin at a much higher frequency than from the posterior margin. Thus initiation from the anterior margin must be able to overcome the inhibition normally caused by wg. wg inhibitory function is shown to act downstream of or in parallel to the action of Thickveins, a receptor for Dpp (Hazelett, 1998).

Rather than affecting the Dpp pathway directly, Wg might block photoreceptor differentiation at a stage subsequent to Dpp signaling. Formation of all photoreceptors is known to depend on the EGF receptor and its downstream component Ras. Wg has recently been shown to antagonize EGF receptor signaling during the specification of the cuticle pattern in the embryo. To determine whether wg also acts on this pathway in the eye, a test was performed to determine if a secreted and active form of the ligand Spitz (s-Spi) or a constitutively active form of Ras could bypass the block caused by wg. In discs expressing both Wg and activated Ras ubiquitously, extensive photoreceptor differentiation and growth are observed, as in discs expressing activated ras alone. Thus Wg must act upstream, prior to Ras activation, to block differentiation. Expression of s-Spi also rescues photoreceptor differentiation in discs expressing Wg ectopically. The inhibition of photoreceptor differentiation is mediated by the conventional Wg signal transduction pathway, because a constitutively active form of Armadillo, a mediator of the Wg signal, is shown to block photoreceptor differentiation (Hazelett, 1998).

Since the phenotype caused by ectopic wg is rescued by expressing activated forms of Spi or Ras it is possible that Wg interferes with Egf receptor signaling upstream of (prior to the activation of) Ras. Recently, it has been shown that in the embryonic segments Wg and secreted Spi emanate from distinct sources and promote opposing cell fates. This led to the proposal that Wg antagonizes signaling by Spi through the EGF receptor and the Ras/MAPK cascade. Since EGF receptor signaling is required for the formation of all photoreceptors, it is a possible target for Wg inhibition in the eye disc. However, it does not appear that the effects of ectopic Wg can be completely explained by the antagonism of Spi signaling, since mutations in spi allow the specification of R8 and the progression of the furrow, while the presence of ectopic Wg does not. It is possible that another ligand, such as Vein, normally activates the EGF receptor in R8 and that this ligand is also antagonized by Wg. Another possibility is that Ras activation in R8 is mediated by another tyrosine kinase receptor; one of the identified FGF receptors is expressed in the morphogenetic furrow. The lower effectiveness of rescue by s-Spi than by activated Ras also suggests that Wg has effects both upstream of Spi expression or processing, and downstream of these events. Some factors known to be required between Spi and Ras that could be targets of Wg inhibition are Daughter of sevenless, Downstream of receptor kinases and Son of sevenless. Alternatively, Wg could act by stimulating the expression or function of Argos, a secreted antagonist of Spi (Hazelett, 1998).

Genes implicated in early events of eye development, eyes absent and eyegone, are involved in the regulation of dpp and wg expression. The expression of dpp and wg were examined in eya mutant eye discs. Expression is greatly reduced in early third instar eya mutant discs, prior to the initiation of the morphogenetic furrow, and was completely lost in eya mutant clones, suggesting that eya is required for dpp transcription. Although the initiation of wg expression in early eya mutant eye discs appeared to be normal, ectopic Wg protein was observed in eya mutant clones in late third instar discs. Another gene required for eye formation that has not been placed within the hierarchy of eye development is eyegone (eyg); in its absence, no photoreceptors differentiate and the eye disc does not reach its normal size and shape. Eyegone is a Pax-like protein (C. Desplan and H. Sun, personal communication to Hazelett, 1998). The expression patterns of dpp and wg were examined in early third instar eyg mutant discs. dpp expression is restricted to the posterior margin of eyg mutant discs, in contrast to its expression around the posterior and lateral margins of wild-type discs. On the contrary, wg expression was expanded, especially on the dorsal side of the disc, where it extends to the posterior margin. eyg thus acts to delimit the domains of dpp and wg expression; since it encodes a Pax-like transcription factor, it is possible that this regulation is direct. Since inhibition of the wg pathway at the posterior margin of eyg mutant discs is sufficient to allow photoreceptor formation, it is concluded that the misexpression of wg observed at the posterior of the eyg mutant discs is a major cause of the absence of photoreceptor development. As expected since it can overcome the effect of ectopic wg, activated Ras is also able to rescue photoreceptor differentiation in eyg mutant discs. In summary, these results show that wg inhibits normal photoreceptor differentiation in a manner independent of dpp expression or activation. The expression patterns of both dpp and wg, and perhaps their cross-regulatory interactions, are determined during early eye development by genes including eya and eyg (Hazelett, 1998).

eyegone (eyg) is required for eye development. Loss-of-function eyg mutations cause reduction or absence of the eye. Similar to the Pax6 eyeless (ey) gene, ectopic expression of eyg induces extra eye formation, but at sites different from those induced by ey. Several lines of evidence suggest that eyg and ey act cooperatively: (1) eyg expression is not regulated by ey, nor does it regulate ey expression; (2) eyg-induced ectopic morphogenetic furrow formation does not require ey, nor does ey-induced ectopic eye production require eyg; (3) eyg and ey can partially substitute for the function of the other, and (4) coexpression of eyg and ey has a synergistic enhancement of ectopic eye formation. These results also show that eyg has two major functions: to promote cell proliferation in the eye disc and to promote eye development through suppression of wg transcription (Jang, 2003).

eyg appears to have two major functions. The first is to promote cell proliferation in the eye disc. eyg loss-of-function mutants have reduced eye discs, already apparent in early third instar, before photoreceptor differentiation. In clonal analysis, eygM3-12 mutant clones induced in first or second instar are undetectable in late third instar eye disc. Ectopic eyg expression causes local overgrowth, a phenotype opposite that of the loss-of-function phenotype. The overgrowth does not always develop into photoreceptor cells. These results indicate that eyg promotes cell proliferation independent of photoreceptor differentiation. The second function of eyg is to promote eye development or MF initiation. If the eyg-induced proliferation occurs at the ventral margin of the eye disc, ectopic MF can initiate. The induction of ectopic MF is probably mediated by the suppression of wg, which is known to repress MF initiation along the lateral margins (Jang, 2003).

Dpp and Wg are two signaling molecules important for the initiation of eye differentiation: Dpp activates MF initiation while Wg suppresses it. Does eyg exert its effect on eye development by activating Dpp signaling or by suppressing Wg signaling? dpp is expressed at two stages in the eye disc: an early expression along the posterior and lateral margins, and a later expression in the propagating MF. The early expression in the margins is required for MF initiation. It was found that dpp expression along the lateral margins is absent in early third instar eyg1 eye disc, suggesting that dpp expression in the lateral margins is regulated by eyg. However, activating DPP signaling at the lateral margin does not rescue the eyg1 phenotype, suggesting that eyg has other functions in addition to activating dpp expression (Jang, 2003).

wg is expressed uniformly in the eye disc of second instar larvae. In the third instar eye disc, wg is expressed in the lateral margins and acts to prevent MF initiation from the lateral margins. The wg-expression domain expands in eyg1 eye discs. The results further show that ectopic eyg expression (dpp>eyg) can suppress wg expression at the transcriptional level. The suppression of wg is functionally significant, because expression of the wg-activated omb gene is similarly suppressed in dpp>eyg. Blocking of the Wg signaling pathway can partially rescue the eyg mutant phenotype. These results indicate that the suppression of wg transcription by eyg may be a major mechanism by which eyg induces MF initiation, hence eye development. This is consistent with the finding that ectopic eyg induces ectopic eye formation primarily in the ventral margin of the eye disc, where wg expression is weaker and most easily suppressed by dpp. wg is normally expressed in the entire eye disc during second instar. It has been shown that Wg signaling can suppress the expression of so and eya. It is possible that in the late second instar eye disc, eyg expression in the central domain of the eye disc suppresses wg expression in the central domain, thus allowing the expression of eya and so, hence eye development (Jang, 2003).

As predicted by the eyg and ey interaction, ey also suppresses wg expression. Suppression of wg expression by eyg (and ey) is also seen in the wing disc. However, suppression does not occur in all cells expressing eyg, suggesting that additional factors are required for the wg suppression. The relationship of eyg/ey and wg may be mutually antagonistic, since ectopic ey cannot induce eya and so expression in regions of high wg expression (Jang, 2003).

Dichaete/fish-hook null mutants are embryonic lethal and exhibit severe defects in segmentation and CNS development. Thus, to directly examine fish postembryonic functions mitotic clones of fish were generated using the fish87 null mutant and the FLP/FRT system. Recombination was induced at different developmental stages and the resulting fish mutant clones were detected in adult animals by morphological inspection using the yellow (y) and white (w) mutations as markers, and in larval tissues by anti-Fish immunostaining. The effects of loss of fish function on gene expression in developing larval tissues were analyzed. In particular, the expression of two key developmental regulators, wingless (wg) and engrailed (en), were examined in fish mutant clones using a P[wg-lacZ] reporter and Mab 4D9. In leg discs, wg is normally expressed in a wedge in the anterior/ventral quadrant. In some individuals where fish mutant clones were induced, wg expression was absent in small patches of cells near the tibial/tarsal boundary. This corresponds to a region of prominent Fish expression in normal leg discs. Thus, fish function is required for wg expression during larval development. The loss of fish function is also associated with defects in en expression. When fish clones were induced, small patches of cells lacking en expression were detected in regions of the antennal disc corresponding to sites of Fish expression. Fish is also normally expressed in many cells in the larval brain, and the loss of fish is associated with an absence of en expression in discrete clusters of brain cells. Taken together, these loss-of-function studies indicate that fish plays an important role in regulating larval gene expression and cell differentiation (Mukherjee, 2000).

The formation of different structures in Drosophila depends on the combined activities of selector genes and signaling pathways. For instance, the antenna requires the selector gene homothorax, which distinguishes between the leg and the antenna and can specify distal antenna if expressed ectopically. Similarly, the eye is formed by a group of 'eye-specifying' genes, among them eyeless, which can direct eye development ectopically. hernandez (distal antenna related or danr) and fernandez (distal antenna or dan) are expressed in the antennal and eye primordia of the eye-antenna imaginal disc (see Dan and Danr). Hernandez and Fernandez are the names of twin brothers in Tintin comic-books. The predicted proteins encoded by these two genes have 27% common amino acids and include a Pipsqueak domain. Reduced expression of either hernandez or fernandez mildly affects antenna and eye development, while the inactivation of both genes partially transforms distal antenna into leg. Ectopic expression of either of the two genes results in two different phenotypes: such expression can form distal antenna, activating genes like homothorax, spineless, and spalt, and can promote eye development and activates eyeless. Reciprocally, eyeless can induce hernandez and fernandez expression, and homothorax and spineless can activate both hernandez and fernandez when ectopically expressed. The formation of eye by these genes seems to require Notch signaling, since both the induction of ectopic eyes and the activation of eyeless by the hernandez gene are suppressed when the Notch function is compromised. These results show that the hernandez and fernandez genes are required for antennal and eye development and are also able to specify eye or antenna ectopically (Suzanne, 2003).

The formation of the morphogenetic furrow in the eye is limited laterally by wg signaling. hern and fer expression within the eye primordium includes the more lateral wg-expressing regions. Interestingly, both hern and fer activate wg transcription when ectopically expressed. In ptc-GAL4/UAS-hern or dpp-GAL4/UAS-fer flies, the wings show several alterations, including the appearance of marginal bristles in the middle of the wing blade. This phenotype is characteristic of ectopic wg signaling, and in fact, wg is ectopically expressed in the wing discs of these larvae. Clones expressing the fer genes in the eye-antenna, leg, or wing discs also show induction of wg, mostly within but also outside the clone. The elav gene is also induced nonautonomously by these clones. Cells ectopically expressing elav do not coincide with those expressing wg and this reproduces the wild-type situation in the eye (Suzanne, 2003).

Transformations are observed of third antennal segment, where hern and fer are normally transcribed, to eye tissue, in Dll-GAL4/UAS-hern or dpp-Gal4/UAS-fer flies. This suggests that the levels of Hern and Fer products may be important in inducing or maintaining ey expression and distinguishing eye from antenna. Accordingly, when Hern or Fer products are increased in the antennal primordium, the expression of hth, an inhibitor of eye development, is eliminated. It is also noted that, in the wild-type eye-antennal discs, hern and fer show higher levels of expression in the eye primordium than in the antennal one, where these genes are coexpressed with hth. However, the amount of Tintin product is not the only factor in this distinction, since, for instance, in Dll-GAL4/UAS-hern eye-antennal discs, the area of ectopic ey transcription in the antenna is smaller than the area of hern overexpression. The activity of other genes will probably contribute to the formation of either eye or antenna. Thus, the ectopic expression of either hern or fer induces wg, an inhibitor of morphogenetic furrow formation, and this probably limits the places where the eye can develop (Suzanne, 2003).

Suppression of Polycomb group proteins by JNK signalling induces wg and vg expression and transdetermination in Drosophila imaginal discs

The PcG proteins function through cis-regulatory elements called PcG response elements (PREs), which enable them to bind and to maintain the state of transcriptional silencing over many cell divisions. PcG proteins operate in two key evolutionarily conserved chromatin complexes, and reduced expression of these complexes, as found in PcG mutants, results in the derepression of PRE-controlled genes. To determine whether PcG silencing is modulated in regenerating tissue, the FLW-1 line, which contains a lacZ reporter gene under the control of the Fab7 PRE, was used. Prothoracic leg discs silent for lacZ expression were fragmented and transplanted into the abdomen of host flies. Flies were fed with 5-bromodeoxyuridine (BrdU) to mark the regenerated tissue (the blastema). In uncut discs, there was little proliferation and expression of lacZ was undetectable. On fragmentation, however, lacZ was expressed in the blastema. To confirm that this derepression was due to a reduction in PcG silencing and not simply to massive proliferation at the wound site, the line LW-1 was used; this line lacks the Fab7 PRE and is normally silent, but it can be activated by induction of GAL4. Neither uncut nor cut leg discs of the LW-1 line showed expression of lacZ after transplantation (Lee, 2005).

To show that transdetermination takes place only in cells with downregulated PcG function, fragmented leg discs of the FLW-1 line were stained for lacZ expression and for Vg in order to visualize the transdetermination to wing fate. It was consistently observed that the Vg staining lay within the lacZ expression domain, suggesting that PcG genes are downregulated in the blastema, enabling PRE-silenced genes to be reactivated according to new morphogenetic cues (Lee, 2005).

To investigate direct targets of PcG regulation that, when reactivated, might contribute to transdetermination, the PREs predicted at the wg and vg genes were tested and both were found to be controlled by PcG proteins. The fact that both the transgenic vg-lacZ reporter construct (which lacks the PRE) and the endogenous vg gene were upregulated in the blastema suggests that PcG proteins may affect vg expression both indirectly (for example, through wg) and directly by means of the vg PRE (Lee, 2005).

JNK signalling in Drosophila is crucial for wound healing and is implicated in many different developmental processes, such as dorsal and thorax closure. hemipterous encodes the JNK kinase (JNKK) that activates the Drosophila JNK Basket. Products of DJun and kayak (the Drosophila homologue of Fos) form the AP-1 transcription factor. A downstream target of JNK signalling is puckered (puc), which encodes a phosphatase that selectively inactivates Basket and thus functions in a negative feedback loop. The expression of puc thus mirrors JNK activity. Because wound healing takes place after fragmentation, it was reasoned that activation of the JNK pathway might be causing the downregulation of PcG proteins in the blastema. The pucE69 line, which carries a P(lacZ) insertion at the puc locus, was used to monitor JNK activity. During the third-instar larval stage puc is not expressed and thus JNK signalling was not activated in leg discs. As expected, however, puc was expressed on fragmentation in all cells at the annealing cut edge (Lee, 2005).

To check whether cells that have activated the JNK pathway also show transdetermination, fragmented leg discs of flies carrying the puc-lacZ reporter and vgBE-Gal4; UAS-GFP constructs were transplanted. In these flies, cells that adopted a wing fate were identified by their expression of green fluorescent protein (GFP). Two days after fragmentation, weak residual puc-lacZ staining was still visible in the central region of the disc. puc-lacZ staining is known to decline rapidly after wound healing is completed. It was found that stronger staining was visible along the cut site, probably owing to ongoing wound healing. On comparison of puc-lacZ staining and GFP fluorescence, JNK-active cells showed a substantial overlap with transdetermined cells; thus, it is concluded that JNK signalling is activated in cells that undergo transdetermination (Lee, 2005).

JNK signalling affects the transcription of numerous genes, including those encoding chromatin regulating factors. Therefore whether JNK signalling can downregulate the PcG proteins required for transdetermination was examined. A constitutively active form of hep was overexpressed in UAS-hepact; hsGal4 flies by a heat-shock pulse. Activating the JNK pathway caused a downregulation of some PcG genes, such as Pc, ph-p and E(Pc). No downregulation of these genes was observed in wild-type larvae before and after heat shock, indicating that this was not an unspecific heat-shock response. Expression was examined of two genes of the Trithorax group (ash1 and brm) that function antagonistically to PcG proteins, but found no upregulation on JNK induction (Lee, 2005).

To show further that JNK has a specific effect on PcG proteins, the analogous experiment was carried out in mammalian cells. The JNK pathway can be activated in mouse embryonic fibroblasts by exposing the cells to ultraviolet light. The expression of MPh2 (mouse polyhomeotic2) was examined because this mammalian PcG gene is expressed in these cells. The expression of MPh2 was decreased on JNK induction, but after treatment with a specific JNK inhibitor it was partially restored. In addition, to show that the downregulation of PcG genes is directly controlled by AP-1, chromatin immunoprecipitation was carried out using antibodies against Fos on chromatin from UAS-hepact; hsG4 and kay1 mutant flies. Enrichment of Fos on the promoter region of ph-p was observed, but no enrichment in chromatin from flies lacking Fos. This finding suggests that AP-1 binds directly to this region to regulate negatively the transcription of ph-p (Lee, 2005).

If activation of JNK signalling in the blastema indeed leads to a downregulation of PcG genes, then impairment of the JNK pathway should result in reduced efficiency of transdetermination. The transdetermination behaviour of wild-type discs was compared with that of discs bearing mutations in the JNKK hep. The transdetermination events were classified into three categories: large regions, small regions, and no regions of transdetermination. In wild-type discs only large regions were detected. In males hemizygous for hep1 (a weak hypomorphic allele), most transplanted leg discs had large transdetermined regions; however, a substantial proportion showed only small regions of transdetermination and a few showed no transdetermination event. In flies heterozygous for hepr75 (a null allele which is hemizygous lethal), most discs showed no or only small regions of transdetermination, and large regions were rarely seen. The morphology of the regenerated discs seemed unaffected in these mutants, indicating that the decline of transdetermination efficiency was not due to inefficient wound healing (Lee, 2005).

This study has shown that PcG genes are downregulated by JNK signalling. Because many developmental regulators need to be switched, the role of PcG downregulation may be to render the cells susceptible to a change in cell identity by shifting the chromatin to a reprogrammable state. Transdetermination has been ascribed to the action of ectopic morphogens, which induce cells to activate incorrect gene cascades. Without doubt, wg and decapentaplegic signalling must be crucially involved in this process, because transdetermination does not result from any random cut but occurs preferentially when cuts are made through particular regions of the disc called 'weak points', which are regions of high morphogen. Inappropriate or overextreme downregulation of the PcG system by JNK in sensitive cells of the weak points thus may create such aberrant local patterns. Indeed, the data indicate that at least the two patterning genes, wg and vg, may be direct targets of the PcG. Notably, hyperactive Wnt signalling can also induce a switch in lineage commitment in mammals, implying that signalling pathways are a potent inducer of cell fate changes in many organisms (Lee, 2005).

Another study has shown that regenerating and transdetermining cells in the blastema have a distinct cell-cycle profile in contrast to the surrounding normal disc cells. It has been proposed that this change in cell-cycle regulation is a prerequisite for the change in cell fate. Indeed, PcG targets include genes involved in cell-cycle regulation, suggesting that this initial step is part of the complete reprogramming cascade required for the regenerating cells to achieve multipotency. Downregulation of PcG silencing by JNK seems to be a fundamental, evolutionarily conserved mechanism of cell fate change and thus may also have implications for studies of stem cell plasticity and tissue remodelling (Lee, 2005).

An antennal-specific role for bowl in repressing wingless and supernumerary appendage development in Drosophila

In Drosophila, antennae and legs are serially homologous appendages, and yet they develop into organs of very different structure and function. This implies that different genetic mechanisms operate onto a common developmental ground state to produce antennae and legs. Still few such mechanisms have been uncovered. During leg development, bowl, a member of the odd-skipped gene family, has been shown to participate in the formation of the leg segmental joints. This study reports that, in the antennal disc, bowl has a dramatically different role: bowl is expressed in the ventral antennal disc to prevent inappropriate expression of wg early during development. The removal of bowl function leads to the activation of wg in the dpp-expressing domain. This ectopic expression of wg, together with dpp, results in a new proximo–distal axis that promotes non-autonomous antennal duplications. The role of bowl in suppressing a supernumerary PD axis is maintained even when the antennal disc is homeotically transformed into a leg-like appendage. Therefore, bowl is part of a genetic program that suppresses the formation of supernumerary appendages specifically in the fly’s head (Brás-Pereira, 2008).

In Drosophila, antennae, mouthparts, legs and genitalia are considered to be serially homologous ventral appendages. This means that despite their very different structure and function, they are thought to develop from a common developmental ground state. It is the segment-specific selector gene expression that, acting upon this ground state, defines their specific morphologies. Of these ventral appendages, the development of the leg is best understood. The leg primordium is set aside as a cluster of epidermal cells, composed of a distal population, that expresses Distal-less (Dll) and a proximal one, expressing homothorax (hth), teashirt and escargot. This early genetic subdivision corresponds to the proximo–distal (PD) telopodite–coxopodite subdivision of the insect appendages. hedgehog (hh), expressed by posterior cells within the leg primordium, triggers the expression of the decapentaplegic (dpp) and wingless (wg) signaling molecules in anterior cells which, through mutual repression, become expressed in a dorsal and a ventral wedge, respectively. wg and dpp expressions only coincide at the center of the leg disc and it is this confluence of maximal signaling that defines the distal tip of the future leg and triggers growth. The larval development of the leg primordium –called leg imaginal disc – progresses by the successive definition of intermediate domains of gene expression that specify the segments of the leg (coxa, trocanter, femur, tibia and tarsus) are defined. During late larval life, leg development becomes wg/dpp-independent, and the distal disc tip becomes a source of EGFR signaling, which is responsible of the further segmentation of the tarsus into the five tarsomeres and the terminal claw. Growth and segmentation of the leg also depends on Notch signaling. Activation of Notch by its ligands Delta (Dl) and Serrate (Ser) is necessary for the disc to grow, and the overlapped expression of Dl and Ser in concentric rings defines the position of the joints of the leg segments as the cells immediately distal to these rings (Brás-Pereira, 2008 and references therein).

The odd-skipped family of genes, odd-skipped (odd), drumstick (drm) and sister of odd and bowl (sob) are among the Notch targets in legs. These genes are expressed in concentric rings at the prospective leg joints, just distal to the Dl/Ser ring domains. A fourth member of the family, brother of odd with entrails limited (bowl), has a more widespread expression pattern. Genetic data indicate that bowl is required for the segmentation of the leg, and that the localized co-expression of the other family members allows (probably in a redundant fashion) the activation of bowl at the prospective joints. Further molecular and genetic experiments show that, at least during embryogenesis, the product of the gene lines blocks bowl function by directly binding to Bowl and preventing its nuclear accumulation. Drm and likely Odd are able to competitively displace Lines from Bowl, thus allowing Bowl to become nuclear and functional (Brás-Pereira, 2008 and references therein).

The distinct antennal development is promoted by the distal maintenance of hth expression in the antennal disc, resulting in the co-expression of hth and Dll. This co-expression selects the antennal fate. Compared to the leg, the antenna is a much shorter appendage, with four segments (antennal (a) segments 1–3, plus a distal arista), and functions in olfaction, through the specialization of its a3 segment. The antennal disc does not develop as an independent disc, like the leg one, but forms part of the eye–antennal disc complex. This disc comprises cells derived from several embryonic head segments and the unsegmented acron. All the cells of the eye–antennal disc complex express the Pax6 genes eyeless (ey) and twin-of-eyeless during first larval stage (L1), but during L2, only the posterior two-thirds of the complex express Pax6 genes, while the anterior third expresses cut (ct). The L2 ct and Pax6 domains correspond to the antennal and eye discs, respectively. The smaller size and fewer segments of the adult antenna when compared to the leg correlate with a different expression of the Dl and Ser ligands in antennal and leg discs. Accordingly, the antennal disc has only two odd-expressing rings, instead of the six present in leg discs. The different control of growth and segmentation in the antenna indicates that there must be mechanisms operating differently in antennal and leg discs (Brás-Pereira, 2008 and references therein).

The fact that bowl has been placed downstream of Notch signal in the elaboration of distal leg patterning prompted a test of whether bowl had any function during antennal development, and if it did, whether it was similar to its role during leg segmentation. The results indicate that, during antennal disc development, bowl has a dramatically different role: bowl is expressed at early stages in the ventral antennal disc, where it prevents inappropriate expression of wg. If bowl is removed, the activation of wg results in non-autonomous antennal duplications. bowl is still required to prevent PD axis duplication in homeotically transformed antennal discs, which indicates that there are genetic differences between head and thorax discs that are selector gene independent (Brás-Pereira, 2008). .

During the development of the antennal disc, bowl has two phases of expression: an early expression in the ventral disc, required to maintain wg repressed, and a later one in concentric rings. Both phases have antennal-specific properties. The early bowl expression and function is unique to the antenna. Its expression in rings associated to prospective joints, which recapitulates the ring expression in leg discs, does not seem required for joint formation in the antenna, contrary to what has been described in the legs. In addition, bowl is still required to repress a ventral supernumerary PD axis even if the antenna has been homeotically transformed into a leg-like appendage by overexpression of the leg selector Antp. All these results indicate that the development of the head structures deriving from the antennal disc depends not only on the activity of selector genes, but also on a cephalic-specific genetic program. Supporting this claim, it was found that the expression of eyg, an antennal-specific marker, is maintained in homeotically transformed antennal discs (Brás-Pereira, 2008).

These cephalic vs. thoracic differences might reflect the very different developmental histories of antennal and leg discs. While each leg disc primordium is formed from cells derived from just two adjacent parasegments (or one embryonic segment), the antennal disc is part of a composite disc, the eye–antennal disc, which forms by the fusion of imaginal primordia derived from several embryonic head segments [the labial, antennal, intercalary, mandibular and maxillary segments plus the unsegmented acron. The coalescence of all these primordia in a single imaginal disc might have required the repression of some domains of gene expression carried along by precursor cells. In this sense bowl might have been recruited to block wg expression in the ventral cells of the antennal disc during the early stages of its development. The lack of bowl at this stage would release wg expression which, in turn and with dpp, would trigger the development of a new appendage (Brás-Pereira, 2008).

The repressive function of bowl might extend to other parts of the eye–antennal eye disc. bowl minus clones in the ventral region of the stem that connects the antennal and eye disc lobes develop autonomously into eye tissue. In contrast to the antennal suppressing function, bowl is required autonomously to repress eye development. This autonomy indicates that either the signals normally operating to spread retinal differentiation in the normal eye are not produced in these ectopic retinal patches, or that the wild type tissue is refractory to these signals. At present, no choice can be made between these two hypotheses. It was noticed, however, that the overexpression of the bowl inhibitor Lines driven by the dpp-GAL4 driver leads to two phenotypic outcomes: antennal duplication or ectopic ventral eyelet. Interestingly, only in one case out of more than 20 discs analyzed these two phenotypes co-occurred. This suggests that the cells in the sensitive region adopt collectively only one two fates, antenna or eye, and that deciding upon one excludes the other. Since wg normally acts by limiting the eye field, eye fate might be blocked in those ventral bowl minusclones derepressing wg. In addition, it is noted that this ct, ey-expressing region is particularly prone to develop into eye upon genetic perturbations. For example, it is this region that is preferentially transformed into eye when hth function is removed or when tsh is ectopically expressed. Perhaps, the unique fact that this region co-expresses antennal and eye determinants makes its fate more ambiguous. In the absence of bowl, hth might tilt the equilibrium towards head capsule or antennal development, while the opposite fate – eye – would be adopted in the presence of tsh and ey. It will be interesting to determine whether functional relationships between bowl and these factors exist to determine specific fates within the eye disc (Brás-Pereira, 2008).

Mechanistically, bowl function seems to lie downstream of hh and dpp. In bowl minus cells associated with an antennal duplication, hh is still expressed and the Hh-coreceptor patched is normally up-regulated in anterior cells abutting the hh-expressing domain, which indicates correct hh-signaling. Accordingly, wg derepression in bowl minus cells occurs closest to the P cells, as expected for a hh target gene. In the embryo, bowl has also been placed downstream of hh during the process of epidermal differentiation (Brás-Pereira, 2008).

In the antenna, as in the leg disc, the dpp and wg signaling pathways repress each other to establish two opposing wedges of dpp and wg expression. In bowl minus clones, though, dpp expression, monitored by a lacZ-expressing reporter, is not turned off, despite the induction of wg expression ventrally. Although this might be due to the perdurance of the LacZ product, bowl minus cells accumulate normal levels of phosphorylated-Mad. This indicates that bowl-mutant cells transduce the dpp signal. Therefore, these results suggest that bowl is required for the mutual repression of wg and dpp in the ventral portion of the antennal disc. Nevertheless, bowl is not sufficient to repress wg in the antenna. Simple explanations for this fact have been ruled out, such as low levels of the induced Bowl protein, or its retention in the cytoplasm. This insufficiency is not due to the inhibition by Lines, because even in the presence of Drm, which prevents Lines from binding to Bowl, this latter is still unable to repress wg. Although further work is required to identify which other factor or factors collaborate with bowl during ventral antennal disc development, the simplest explanation would be that Bowl acts in concert with a factor induced by dpp. This is because bowl cannot block the ectopic wg expression in ventral antennal cells devoid of dpp signal. Nevertheless, when bowl expression is forced in the leg disc using the ptc-GAL4 driver, wg is repressed by bowl cell-autonomously in the most distal region of the disc, but not in the more proximal domain. This result strengthens the idea that bowl acts as a wg repressor. Such repression takes place in the distal part of its domain, closest to the dpp source, which also supports the claim that bowl requires the dpp signaling to repress wg (Brás-Pereira, 2008).

This study has shown that bowl is expressed in the ventral antennal disc, the realm of the dpp pathway, and that dpp signaling can activate bowl transcription in this disc. These results suggest that high levels of dpp induce bowl which, in turn, is required to prevent inappropriate expression of wg in the antennal disc together with the dpp pathway. Two are the likely sources of Dpp: the wedge of dpp that can be visualized using the dpp-disc enhancer reporters in the antenna, and a ventral disc expression that is controlled by a separate enhancer. This enhancer drives dpp expression in the prospective ventral head region, close to the region where bowl is transcribed in early discs (Brás-Pereira, 2008).

bowl and the related genes odd and drm show a late pattern of expression in rings, similar to the one deployed in leg discs. But contrary to their requirement for leg segmentation, bowl seems to be dispensable for antennal segmentation. A similar situation has been described for the gene dachshund (dac). dac is expressed in the medial segment of both leg and antennal discs, but while loss of dac in the leg leads to the loss of intermediate adult leg structures, the antenna develops normally. These results might reflect the fact that, although antennal and leg discs have specific developmental programs, the mechanisms for generating the PD axis are shared by both appendages. This mechanism would call a similar battery of genes, even if only a subset of them is effectively used for the development of each appendage. In fact, ectopic activation of the Notch pathway by overexpression of the ligand Delta induces ectopic expression of drm in the antenna. This indicates that, similarly to what happens in the leg discs, ring expression of Odd-family genes in the antenna might also be under Notch control. In this sense, in the antenna the segmentation function might have been taken over by other(s) member(s) of the Odd family, expressed as well in the future joints (Brás-Pereira, 2008).

In summary, the results show that the zinc-finger encoding gene bowl is part of a cephalic-specific program that represses appendage formation in the ventral eye–antennal disc. Here, bowl is required to repress wg, downstream of dpp, to prevent the generation of supernumerary antennae. These extra appendages might arise from some silenced primordium in the proximal part of the antenna, which would be normally fated to become part of the head capsule. In addition, bowl also silences the development into eye of another cell population of the prospective head that presents mixed expression of antenna and eye selector genes. The repressive action of bowl that is described here might have been essential for the coalescence of cells deriving from several different embryonic cephalic segments into a single imaginal disc, as well as for the formation of the head structures of adult cyclorraphan flies, such as Drosophila (Brás-Pereira, 2008).

Wingless expression in gut

Segment polarity genes are not activated in the anterior by pair-rule genes, as they are in the trunk but instead are activated by gap genes. The segment polarity genes hedgehog and wingless are two important targets of cnc and forkhead, expressed in the anterior and posterior gut anlagen. cnc is expressed in the labial region of the foregut, fated to give rise to the dorsal pharynx. fkh is expressed in the adjacent esophagus. fkh is responsible for the maintenance but not the initiation of wg synthesis in the invaginating esophageal primordium. cnc is responsible for the maintenance of wg in the dorsal pharyngeal domain of wg expression. Expression of hedgehog is similarly affected in cnc and fkh mutants. It is not known whether the actions of cnc and fkh on hh and wg are direct or indirect (Mohler, 1995a). Expression patterns of wg, teashirt and decapentaplegic are altered in the embryonic midgut of embryos lacking exd, while the expression of their respective regulators (abd-A, Antp and Ubx) remains normal. (Rauskolb, 1994).

forkhead is required for the activation of wingless, hedgehog and decapentaplegic in both the foregut and hindgut, considered to be ectodermal tissues. wingless is expressed initially in the whole hindgut primordium, but becomes restricted to a ring in the small intestine anterior to the outgrowing Malpighian tubules, and to a ring in the posterior region of the rectum. hedgehog is also expressed in the hindgut primordium but becomes restricted to a ring of cells posterior to the outgrowing Malpighian tubules in the future small intestine of the hindgut. A second hh expression domain is located in the anterior portion of the rectum. These two expression domains are adjacent to the wg expression domains. dpp is expressed in the hindgut primordium and later on one side in the large intestine of the hindgut tube, in between the small intestine and the rectum. Thus the expression domains of wg, hh and dpp subdivide the hindgut tube into a central portion (the large intestine) where dpp is expressed, and two flanking regions (the small intestine and the rectum) where wg and hh are expressed. In fkh mutant embryos, the foregut, the midgut and the hindgut epithelia are disrupted, and fkh is required for the activation of each of these genes in the fore- and hindgut primordia. fkh is expressed in the entire foregut and hindgut, whereas wg, hh and dpp are expressed only in restricted domains. Since the expression of these genes appear not to be established through cross-regulatory interactions, there must be other factors which act to spatially regulate wg, hh and dpp expression along the hindgut (Hoch, 1996).

Hox genes have large expression domains, yet these genes control the formation of fine pattern elements at specific locations. The mechanism underlying subdivision of the abdominal-A (abdA) Hox domain in the visceral mesoderm has been examined. AbdA directs formation of an embryonic midgut constriction at a precise location within the broad and uniform abdA expression domain. The constriction divides the abdA domain of the midgut into two chambers, the anterior one producing the Pointed (Pnt) ETS transcription factors and the posterior one the Odd-paired (Opa) zinc finger protein. Transcription of both pnt and opa is activated by abdA. Near the anterior limit of the abdA domain, two signals, Decapentaplegic and Wingless, are produced, in adjacent non-overlapping patterns, under Hox control in mesoderm cells. AbdA is proposed to activate three targets, in distinct subsets of its broad domain of expression: wg at the anterior boundary of Connectin (Con) patch 7; pnt from anterior Con patch 7 to anterior Con patch 8, and opa, from anterior Con patch 8 through Con patch 11. Dpp signaling plays a central role in setting these distinct expression domains. The initial activation of wg by AbdA requires dpp. opa is activated in all abdA-expressing cells that do not receive a Dpp signal, defining the site of the posterior constriction. wg, in collaboration with abdA, activates pnt to generate the appropriate number of cells in the third midgut chamber, positioning the posterior constriction at the proper distance from the central constriction and partitioning the posterior midgut appropriately. Fine patterning of the posterior midgut is achieved by the activity of diffusible signals emanating from the central midgut, a remarkably long-range organizing effect (Bilder, 1998a).

The subdivision of the lateral mesoderm into a visceral (splanchnic) and a somatic layer is a crucial event during early mesoderm development in both arthropod and vertebrate embryos. In Drosophila, this subdivision leads to the differential development of gut musculature versus body wall musculature. biniou, the sole Drosophila representative of the FoxF subfamily of forkhead domain genes, has a key role in the development of the visceral mesoderm and the derived gut musculature (Zaffran, 2001).

Besides tissue-specific differentiation genes that are expressed throughout the trunk visceral mesoderm, several key regulators of midgut morphogenesis are known to be expressed in a spatially restricted manner within this tissue. This type of gene product includes the homeotic factor Ubx and the secreted factor Dpp, both of which are expressed in PS7 of the visceral mesoderm. Although it has been established that Ubx and Dpp maintain their expression in PS7 through a crossregulatory loop and the action of Wg from the adjacent PS8, there is evidence that their expression requires at least one additional, visceral mesoderm-specific cofactor, for which Bin may be a candidate. To test this possibility, Ubx and dpp expression were examined in bin mutant embryos, which carried bap3-lacZ, to allow the unambiguous identification of the disrupted visceral mesoderm layer. Visceral mesoderm expression of Ubx in bin mutant embryos is similar to that of wild-type embryos until at least stage 13, although there is a low level of ectopic expression. Likewise, Ubx expression is also observed in ß-gal-positive cells in bap mutant embryos, albeit with reduced levels and an expanded domain: These conditions are comparable to those in the somatic mesoderm. These data demonstrate that the establishment of Ubx expression in the visceral mesoderm requires neither bin nor bap activity. In contrast, dpp is not expressed at any stage in PS7 in the visceral mesoderm of bin mutant embryos, indicating that Bin may serve as a critical tissue-specific cofactor for the regulation of dpp expression. The expression of wg in PS8 is also absolutely dependent on bin activity. The absence of these morphogenetic factors is likely to contribute to the defective midgut morphology in bin mutant embryos (Zaffran, 2001).

Wingless involvement in heart development

even-skipped is also expressed in heart precursor cells in the mesoderm, and is involved in the process of mesodermal segmentation. Expression of eve depends on Wingless, supplied either endogenously from mesodermal cells, or exogenously, from overlying ectodermal cells. even-skipped is expressed in clusters even when wingless is uniformly expressed, suggesting that Wingless is acting here in a permissive and not in an instructive role (Lawrence, 1995).

Additional evidence that wg is directly involved in heart development comes from an analysis of hedgehog function in heart morphogenesis. hedgehog is known to positively regulate wg in ectodermal segmentation, and hh null mutants lack most eve expressing heart precursors. wg acts epistatically to hh, as overexpression of wg corrects for the lack of heart cells in hh mutants. Conversely, overexpression of hh does not correct for a wg deficiency. dishevelled is required for the wingless function in heart formation, since overexpression of dsh in wg mutant embryos restores heart formation. Likewise, armadillo is required for wg signaling in heart formation, as arm mutant embryos completely fail to form heart precursors. zeste white 3 (shaggy) which is known to act antagonistally to wg in segmentation, may not be involved in wg-dependent heart development (Park, 1996).

Transcriptional Regulation Table of contents

wingless continued: Biological Overview | Evolutionary Homologs | Targets of Activity | Protein Interactions | mRNA Transport | Developmental Biology | Effects of Mutation | References

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