Transcriptional Regulation

Rhomboid plays a role in processing of the Gurken signal during oogenesis. The zinc finger transcription factor CF2 is a mediator of EGF-R activated dorsoventral patterning in Drosophila oogenesis. Dorsal ventral polarity is established by the Gurken signal from the oocyte to the anterior dorsal follicle cells which uses the EGF receptor pathway. CF2 is suppressed by EGF-R signaling in the anterior dorsal domain of the follicular epithelium where the dorsal signal is received. In turn CF2 depletion expands the expression of dorsal genes such as rhomboid. Thus CF2 is a candidate for regulation of rhomboid in follicle cells (Hsu, 1996).

Dorsoventral (D/V) patterning in Drosophila oogenesis is initiated by the transmission of a TGF-alpha-like ligand, Gurken (Grk), from the oocyte to the anterodorsal follicle cells, activating the EGF receptor (Egfr) signaling pathway. The zinc-finger transcription factor CF2 is a negative regulator of the rhomboid (rho) gene that encodes an essential membrane-bound component of the dorsalizing pathway. Expression of CF2 itself is negatively regulated by the activated Egfr. CF2 is the target of down-regulation by the MAPK kinase cascade; this down-regulation is independent of the Rho function. These results suggest that D/V patterning involves a two-step signaling process: the initial Egfr signal (which represses CF2 and induces rho expression), and the subsequent Egfr + Rho signal, which determines the dorsal cell fates. CF2 down-regulation occurs at the post-translational level through a mechanism involving coupled cytoplasmic retention and degradation (Mantrova, 1998).

Among stage 10-12 wild-type egg chambers examined for CF2 protein expression, about 10% show elevated cytoplasmic levels of CF2 in the anterodorsal region. This pattern is particularly striking when the elevated cytoplasmic level of CF2 is contrasted to the empty anterodorsal nuclei. Such a pattern indicates that the Egfr signaling cascade may negatively regulate CF2 function by controlling its subcellular localization. In support of this hypothesis, cytoplasmic accumulation of CF2 can be enhanced in the presence of ectopically expressed constitutively active Raf. In addition, CF2 is presumed to be subject to rapid degradation, as most of the wild-type egg chambers show a complete elimination of CF2 protein in the anterodorsal follicle cells. This mechanism is just the opposite of the one at work in Dorsal protein regulation; Dorsal protein function is activated by translocating into the nucleus and the protein is stable throughout its steady state in the cytoplasm (Mantrova, 1998).

Examination of the CF2 amino acid sequence reveals one optimal consensus MAPK phosphorylation site at residue 40, where the threonine residue is presumed to be the phosphorylation target. The importance of the MAPK site was tested by site-specific mutagenesis, quantifying the effect in cultured cells. The A40 mutation does not by itself alter the stability of CF2 in the absence of activated MAPK, at least when its half-life is measured in the S2 cells. Three types of subcellular localization of CF2 are observed; nuclear (N), cytoplasmic (C), and both (N+C). When expressed alone from an actin 5c gene promoter, CF2wt protein is localized to the nucleus in over 92% of all CF2-expressing cells. In the presence of either Ras1V12 or MAPKSem, there is a significant increase in cytoplasmic accumulation of CF2wt: the percentages of cytoplasmic CF2-containing cells (N + C and C) increase to 44% with Ras1V12 and 52% with MAPKSem. In contrast, the percentages of cells expressing cytoplasmic CF2A40 mutant protein remain largely unchanged at the background level in the presence of Ras1V12 or MAPKSem (Mantrova, 1998).

It is proposed that the Egfr-mediated D/V patterning is a two-step signaling process, demarcated by the appearance of Rho function. This model is based on three previous observations: (1) down-regulation of CF2 precedes rho expression; (2) overexpression of CF2 can suppress rho expression; and (3) Egfr alone cannot induce dorsal cell fates without Rho. In this report, the Ras/MAPK signaling pathway has been placed upstream of CF2. But what are the changes in the signaling cascade, if any, brought on by Rhomboid? It has been shown that Rho by itself cannot induce dorsal fates without Egfr, but it can dorsalize the egg chambers when ectopically expressed in the ventral follicle cells, despite the fact that Egfr in the ventral follicle cells is not pre-activated by Grk. Interestingly, the level of CF2 in the ventral follicle cells is not affected by ectopically expressed Rho. This indicates that the signal induced by Rho is at least different from that of Egfr alone, with respect to CF2 regulation. It has been suggested that Rho is involved in processing a second ligand. If this is correct, then Rho may induce a signaling cascade distinct from Ras-Raf-MEK-MAPK, or may modify the specificity of the existing cascade. Indirect evidence has emerged recently that one or more signaling pathway(s) parallel to that of Egfr may in fact exist. Resolving the events downstream of Rho should be the next step in unraveling this complex developmental signaling process (Mantrova, 1998 and references).

During oogenesis, Egfr activation is required for the establishment of the dorsal/ventral axis of the egg and the embryo. To examine how ectopic Egfr activation affects cell fate determination, an activated version of the protein was constructed. Expression of this activated form (lambda top) in the follicle cells of the ovary induces dorsal cell fates in both the follicular epithelium and the embryo. Different levels of expression result in different dorsal follicle cell fates. Among the anterior follicle cells, a minimum of three cell fates can be distinguished by their contribution to the final eggshell morphology. The most dorsal cells produce the midline/operculum region; dorsolateral cells secrete the respiratory appendages, and the ventral cells contribute to the main body. The three populations of follicle cells in the anterior of the developing egg chamber express different combinations of downstream genes. The dorsal midline cells express argos, kekkon1, rhomboid and pointed. The dorsolateral cells express rho and kek1. The ventral follicle cells are distinguished by the expression of CF2 (Queenan, 1997).

Even in cases where all the follicle cells covering the oocyte express lambda top, dorsal cell fates are expanded in the anterior, but not the posterior, of the egg. The expression of genes known to respond to Egfr activation (aos, kek 1 and rho) are also expanded in the presence of the lambda top construct. When lambda top is expressed in all the follicle cells covering the oocyte, kek 1 and argos expression are induced in follicle cells all along the anterior/posterior axis of the egg chamber. In contrast, rho RNA expression is only activated in the anterior of the egg chamber. These data indicate that the response to Egfr signaling is regulated by an anterior/posterior prepattern in the follicle cells. Expression of lambda top in the entire follicular epithelium results in an embryo dorsalized along the entire anterior/posterior axis. Expression of lambda top in anterior or posterior subpopulations of follicle cells results in regionally autonomous dorsalization of the embryos. This result indicates that subpopulations of follicle cells along the anterior/posterior axis can respond to Top/Egfr activation independently of one another (Queenan, 1997).

During Drosophila oogenesis, Gurken, a protein associated with the oocyte nucleus, activates the Drosophila EGF receptor in the follicular epithelium. Gurken first specifies posterior follicle cells, which in turn signal back to the oocyte to induce the migration of the oocyte nucleus from a posterior to an anterior-dorsal position. From this location Gurken signals again to specify dorsal follicle cells, which give rise to dorsal chorion structures, including the dorsal appendages. If Gurken signaling is delayed and starts after stage 6 of oogenesis, the nucleus remains at the posterior pole of the oocyte. Eggs develop with a posterior ring of dorsal appendage material that is produced by main-body follicle cells expressing the gene Broad-Complex. They encircle terminal follicle cells expressing variable amounts of the TGFbeta homolog, decapentaplegic. By ectopically expressing decapentaplegic and using clonal analysis with Mothers against dpp, it has been shown that Decapentaplegic signaling is required for Broad-Complex expression. Thus, the specification and positioning of dorsal appendages along the anterior-posterior axis depends on the intersection of both Gurken and Decapentaplegic signaling. This intersection also induces rhomboid expression and thereby initiates the positive feedback loop of EGF receptor activation, which positions the dorsal appendages along the dorsal-ventral egg axis (Peri, 2000).

The finding that rho expression in the follicular epithelium cannot be induced by Grk alone, but also requires Dpp, shows that both cell-fate specification and cell-fate patterning are controlled by the intersection of the two pathways. Loss-of-function clones have been used to demonstrate that rho and spi are not required for dorsal appendage formation per se but that they are necessary to separate the two appendages and to position them dorsolaterally. Since this patterning mechanism involves the self-amplification of Egfr activation and includes the diffusible ligand Spi, the process must be under tight spatial control to prevent runaway activation in the follicular epithelium. While the localization of Grk limits the process along the DV axis, it is proposed that a Dpp gradient emanating from anterior-terminal cells prevents spreading of the Grk signal along the AP axis of the main-body follicle cells (Peri, 2000).

Hox genes differentially regulate Serrate to generate segment-specific structures: Serrate promotes denticle diversity by precisely localizing a single cell stripe of rhomboid expression

Diversification of Drosophila segmental morphologies requires the function of Hox transcription factors. However, little information is available that describes pathways through which Hox activities effect the discrete cellular changes that diversify segmental architecture. Serrate is a Hox gene target. Serrate acts in many segments as a component of such pathways. In the embryonic epidermis, Serrate is required for morphogenesis of normal abdominal denticle belts and maxillary mouth hooks, both Hox-dependent structures. The Hox genes Ultrabithorax and abdominal-A are required to activate an early stripe of Serrate transcription in abdominal segments. In the abdominal epidermis, Serrate promotes denticle diversity by precisely localizing a single cell stripe of rhomboid expression, which generates a source of EGF signal that is not produced in thoracic epidermis. In the head, Deformed is required to activate Serrate transcription in the maxillary segment, a region where Serrate is required for normal mouth hook morphogenesis. However, Serrate does not require rhomboid function in the maxillary segment, suggesting that the Hox-Serrate pathway to segment-specific morphogenesis can be linked to more than one downstream function (Wiellette, 1999).

Ser transcripts in the trunk are first detected at the extended germband stage in ventral patches in the middle of abdominal segments A2-A8 and in offset lateral patches. The ventral regions of thoracic segments do not exhibit Ser expression at this stage. As the germband retracts, the abdominal stripes intensify and develop sharp anterior borders. The first abdominal segment (A1) is unique: Ser expression begins later than in the other abdominal segments and forms a narrower stripe after germband retraction. After germband retraction, Ser transcripts can also be detected in the ventral regions of thoracic segments in broad, faint patches. Embryos mutant in all genes of the Bithorax Complex (BX-C), Ubx, abd-A and Abdominal-B (Abd-B), develop thoracic-type denticles throughout the trunk region. Consistent with this transformation, stage 11 and 12 BX-C mutant embryos have no Ser expression in ventral regions. Ventral Ser expression does begin in BX-C mutants after germband retraction, but the location and level of expression matches that of the thoracic segments. As expected, Ubx mutant embryos show a transformation of abdominal- to thoracic-type Ser expression only in A1; abd-A mutants show Ser transcript stripes in A2-A8 that are similar to the wild-type A1 pattern, and Abd-B mutants display no change in A1-A8 ventral Ser transcription. Thus Ubx function is sufficient to activate some Ser expression in the center of each segment, but abd-A function is required for the earlier, broader pattern of Ser transcription in A2-A8, a transcript pattern that correlates with complete diversification of denticle belts. Embryos lacking all trunk Hox functions express Ser at the margins of the anterior part of each trunk segment and at lower levels in the center of this region, a pattern almost the inverse of that seen in wild type. Transcription of Ser in the posterior-most region of each segment, probably corresponding to the posterior compartment, is completely suppressed. The delimitation of Ser expression to reiterated subsegmental stripes in the embryonic metameres suggests that segment polarity genes also regulate the Ser transcript pattern. ptc mutants lack ventral abdominal Ser transcripts, correlating with the loss of denticle diversity and number in ptc denticle belts. Ser transcription in wingless (wg) mutants appears in broad stripes, while hedgehog (hh) and engrailed (en) mutant embryos exhibit Ser transcription throughout almost the entire ventral epidermis of the abdominal segments. Broadened patterns of Ser transcription in these segment polarity mutants correspond to expanded fields of denticles that lack significant diversity of denticle type (Wiellette, 1999).

Serrate expression in segments is reflective of its function. The segmental boundary is persistently identified by engrailed expression in the posterior compartment of each segment, and, after germband retraction, by rho in the two most anterior cell rows of A2-A8. By stage 14, Ser transcripts are expressed in two to three rows of cells directly posterior to rhomboid-lacZ expression. The final position of Ser relative to En and rho-lacZ suggests that Ser is expressed in the cells that will produce denticle row 4 and those to the posterior. The anterior boundary of Ser expression appears to lie between the two rows of denticles that Ser affects phenotypically, suggesting that the function of Ser is limited to cells near its anterior boundary of expression. No changes of denticle pattern are found in fringe mutant embryos, indicating that fringe is not required to restrict Ser morphological function in the abdominal segments (Wiellette, 1999).

To understand how Ser affects denticle belt patterning at the single cell level, interactions of Ser with other genes that affect denticle diversity were investigated. In particular, the expression pattern of rho at the anterior of the Ser expression pattern made it a candidate for genetic interaction with Ser. The spitz-group gene rho is required for normal development of abdominal denticle rows 1 and 4. rho transcription first appears in a segmental pattern during stage 12, when it is activated in the anterior cells of each segment. Thoracic segments express rho in one row of cells at the anterior border of each segment, while ventral regions of segments A2-A8 express rho in two rows of cells, the posterior of which is dependent on Ubx/abd-A function. A1 develops rho expression in an intermediate pattern one to two cells wide. In a Ser null background, the abdomen-specific row of rho expression is missing and rho is transformed to the thoracic pattern in all abdominal segments. Stage 13 and early stage 14 Ser mutants show a row of unlabeled cells between Ser expression and the single row of rho expression, demonstrating that it is the posterior row of rho expression that is dependent on Ser. Lower levels of rho expression in Ser mutants suggest that anterior rho is partially dependent on Ser function, perhaps through the intervening posterior rho row. By late stage 14 in Ser5A29 mutant embryos, Ser transcripts have expanded anteriorly and are juxtaposed to the single row of rho. Consistent with this, Ser transcription is detected in an additional anterior row of cells in rho mutant embryos. Thus Ser is required for activation of rho in the posterior row of cells in the abdominal epidermis. Subsequently, rho is required to repress Ser within these same cells (Wiellette, 1999).

Denticle phenotypes show similarities between Ser and rho consistent with the observed regulatory interactions. A wild-type embryo produces six rows of denticles with each row identifiable by polarity and/or size. Ser mutants fail to separate rows 3 and 4, leaving five rows of denticles overall. In rho mutants, individual denticle rows are not as well defined, but it is possible to identify row 5 denticles in the middle of mutant denticle belts. Anterior to row 5 is a row that consists of small, stubby denticles. The most anterior row of denticles in the rho mutants contains sparse row 2 denticles. In rho,Ser double mutants, all of the denticles are similar to each other, and resemble a composite of type 5 and 2 denticles. The denticles are also disorganized, so that separate rows are not distinguishable. The difference between rho and rho,Ser double mutant phenotypes shows that the two anterior denticle rows in a rho mutant are still dependent on Ser for their diverse structures. This suggests that Ser function partially determines anterior denticle identity both within and anterior to its expression domain, independent of rho function. Additionally, the differences between Ser and rho,Ser double mutants show that a single row of rho expression is only partially sufficient to generate denticle diversity to its posterior. Thus the diversity of denticle rows 3 and 4 is dependent both on Ser regulation of rho and on independent Ser functions in denticle rows 3 and 4. Transcription of rho in maxillary segment cells is unchanged in Ser mutants, a result that correlates with wild-type mouth hooks in rho mutants and with the observation that rho,Ser and Ser mutants have identical mouth hook defects (Wiellette, 1999).

A model is presented for the roles of Ser, rho and Hox genes in the generation of denticle belt patterns in the thorax and abdomen. Three Hox genes (Antp, Ubx, and abdA) serve to establish the segmentally specific levels of Ser expression in the third abdominal segment and in the first two thoracic segments respectively. Ser is activated at stage 11 in abdominal parasegments by Ubx and abd-A functions but not in thoracic parasegments where Antp is the principal Hox function. Ubx function is required for the A1-type abdominal expression pattern of Ser, which is narrower and fainter than the pattern in other abdominal segments. This pattern correlates with a narrower, less complex denticle pattern in A1 than in more posterior segments. abd-A function is required for the wider, more abundant Ser stripes in A2-A8. Expression of Ser in the embryonic epidermis results in context-dependent responses, including rho expression, denticle belt patterning and normal development of the mouth hooks. These embryonic roles of Ser are apparently different from its roles in wing margin determination and wing outgrowth. One similarity is the short range over which Ser function is exerted, either at the anterior border of its ventral A2-A8 expression pattern, or at the dorsal/ventral margin of its expression boundary in the wing pouch. The spitz-group gene rho can potentiate Egfr activation via the Spitz (Spi) ligand. Egfr activation is required from late stage 11 to early stage 13 for patterning of the denticle belts, and rho, unlike spi and Egfr, has a spatially and temporally regulated expression pattern. Abdomen-specific rho expression is required for patterning of abdominal denticle rows 1 through 4, probably by allowing secretion of Spitz protein from denticle row 2 and 3 cells, which activates Egfr in neighboring cells. Ser function is required for activation of the abdomen-specific posterior row of rho transcription, expression of which is also dependent on Ubx/abd-A. The evidence presented in this paper suggests that Ser provides a critical intermediate that translates broad Hox and segment polarity domains into narrow stripes of rho expression, which then specify diversification at the single cell level. Ser and rho mutants each show only a single row of denticles between rows 2 and 5 of A2-A8, indicating that Ser and rho are both required for normal development of rows 3 and 4. rho,Ser double mutants develop row 5-like denticle identities throughout the denticle belt. Thus, either gene alone provides some A/P denticle diversity, while the double mutant lacks any diversity. If the only role of Ser were regulation of rho in denticle row 3 cells, then rho,Ser mutants should develop the same phenotype as rho mutants. Since this is not observed, it is concluded that Ser has identity functions independent of rho regulation. Ser function is required in the cells immediately to its anterior expression boundary and within the most anterior row of Ser-expressing cells; the effect within its own domain of expression may be a result of signaling from cells within the same row, or from those to the posterior (Wiellette, 1999 and references).

Ser function, presumably signaling through Notch, is required to determine the identity of the denticle row 3 cells, including activation of rho expression. Segmental rho, in turn, is one of the gene products required for localized Egfr activation in denticle row 4 cells. Thus denticle row 3 identity is dependent on Ser signaling and Rho function, while denticle row 4 identity depends on Ser function and on feedback from rho-expressing cells. This series of events suggests that abdominal Hox functions direct cellular diversification through the establishment of signaling centers. The limitation of Ser function to its anterior border generates a novel boundary within each segment. Activation of a single row of rho expression at this boundary then creates an additional signaling center in A2-A8, which controls the greater morphological diversity in A2-A8 epidermis. This use of a Notch ligand to produce a single cell signaling stripe may be a common theme: in dorsal/ventral wing margin patterning, Notch is activated in a single row of cells on either side of the margin in response to Delta and Ser boundaries, and Notch activation leads to a narrow, Wg-expressing, signaling center. Regulation of rho by the Notch pathway has been demonstrated in Drosophila wing vein patterning. However, in the larval and pupal wing primordia, Delta-activated Notch signaling results in repression of rho expression outside the forming vein. The difference in responsiveness of cells in the wing and the embryonic epidermis could be due to the identity of the signal (Ser versus Delta protein), or to additional identity factors present in the cell. rho, in turn, is required to maintain Delta expression in the provein region. Regulation of Notch function by Egfr activity has been described in C. elegans: translation of a C. elegans Notch homolog is downregulated in response to EGF signaling. Similar regulation may result from rho expression in the Drosophila embryonic epidermis, generating a feedback loop between Ser and rho as has been observed for Dl and rho in the forming wing (Wiellette, 1999 and references).

Wingless and Hedgehog pattern Drosophila denticle belts by regulating the production of short-range signals: Regulation of rho expression

The secreted proteins Wingless and Hedgehog are essential to the elaboration of the denticle pattern in the epidermis of Drosophila embryos. Signaling by Wingless and Hedgehog regulates the expression of veinlet (rhomboid) and Serrate, two genes expressed in prospective denticle belts. Thus, Serrate and veinlet (rhom) partake in the last layer of the segmentation cascade. Ultimately, Wingless, Hedgehog, Veinlet (an indirect activator of the Egfr) and Serrate (an activator of Notch) are expressed in non-overlapping narrow stripes. The interface between any two stripes allows a reliable prediction of individual denticle types and polarity, suggesting that contact-dependent signaling modulates individual cell fates. Attributes of a morphogen can be ascribed to Hedgehog in this system. However, no single morphogen organizes the whole denticle pattern (Alexandre, 1999).

Both Wingless and Hedgehog signaling pathways repress Serrate expression. Since both pathways are believed to activate transcription, it is imagined that they activate the expression of a repressor of Serrate. In addition, Serrate may also be negatively regulated by the transcriptional repressor Engrailed. In contrast to Serrate, veinlet is regulated both positively and negatively: it is repressed by Wingless and activated by Hedgehog. In addition to this vertical flow of information, regulatory interactions also exist between veinlet and Serrate. At the least, Serrate activates veinlet expression by way of the Notch pathway. This effect is purely non-cell autonomous. In contrast, Serrate appears to repress veinlet in a cell autonomous manner (indeed, in cells where it is expressed, Serrate represses the Notch pathway). However, it is also possible that whichever mechanism activates Serrate expression also represses veinlet expression. This would explain why the expression of Serrate and veinlet is always mutually exclusive (Alexandre, 1999).

The regulatory interactions summarized above are sufficient to explain the spatial pattern of both Serrate and veinlet expression. Non-autonomous repression of Serrate by Wingless and Hedgehog ensures that Serrate is expressed in stripes. The spread of Wingless toward the anterior defines the posterior edge of the domain of Serrate expression. Similarly, the anterior edge of the Serrate domain appears to be specified over three cell diameters by Hedgehog slightly further than expected since Hedgehog is thought to act only over 1-2 cells in Drosophila embryos. Expression of veinlet is activated by two different signals, Hedgehog at the anterior and Serrate at the posterior. Although Hedgehog signaling is symmetrical, it does not activate veinlet (rhom) expression anteriorly because there, Wingless represses veinlet expression. Likewise, Serrate activates veinlet expression but only on one side because of unilateral repression by Wingless (Alexandre, 1999).

These interactions display a clear temporal hierarchy. The secreted molecules Hedgehog and Wingless are expressed first and where they do not reach, Serrate expression is subsequently allowed. At stage 11, Hedgehog and Serrate activates veinlet expression in separate cells. Ultimately, this chain of interactions results in detailed patterns of gene expression (Alexandre, 1999).

Mapping the expression pattern of various genes onto the denticle pattern suggests simple correlations. These correlations have allowed the visualization of pattern where it was previously thought there was none, as in wingless mutants. It is now believed that wingless mutants make denticle type 3, 4 and 5 and not exclusively type 5 as has been suggested. The correlations provide a guide to understand various phenotypes such as those of patched mutants and wg-en-double mutants. In wg-en-double mutants, the correlation between gene expression and denticle type/polarity is particularly evident. Expression of veinlet is in circles surrounded by Serrate expression; this correlates with polarity reversal in the cuticle. Non-uniform gene expression shows that these embryos have more pattern than previously noted. For such embryos to be truly unpatterned, they would have to express Serrate uniformly as well as not express veinlet (rhom). This may occur in wg-en-hh- triple mutants since they may not contain any repressor of Serrate. It is presumed that the converse situation (Serrate 'off'and veinlet 'on' everywhere) would also lead to unpatterned embryos. This situation would prevail in wg-ptc-en-triple mutants. Although the correlations have good predictive value, they suffer from several limitations. (1) Denticle shape does not necessarily reflect an integer value. Indeed, unambiguous typing is not always possible and exact denticle shapes vary from segment to segment. (2) Causal relationships between the activation of a particular signaling pathway and a given denticle type still remain to be investigated. The various signaling pathways are predicted to control cytoskeletal behavior, which in turn affects denticle shape and cell polarity. Local polarity reversals indicate that individual cells are able to locate the source of a particular signal, suggesting that subcellular signaling complexes control the cytoskeleton directly. (3) The involvement of additional regulators cannot be excluded. In particular, it is possible that redundant regulators of the Notch and Egfr pathway contribute to the choice of denticle type. These could include Vein (another Egfr ligand), Delta (a Notch ligand) or possibly Fringe. vein is not required for embryogenesis suggesting that it does not play an important role if any. Possible contributions from Delta to denticle patterning are not readily assessed because of Delta's earlier action in neurogenesis (Alexandre, 1999).

These results show that no single morphogen organizes the denticle pattern: patterning arises, at least initially, from the combined actions of Wingless and Hedgehog. Wingless is clearly not involved in the specification of denticle types (or diversity) across each belt since it does not act in this region of the epidermis. If it did, veinlet and Serrate would not be expressed because, as has been shown, they are both repressed by Wingless. Nevertheless, Wingless acts at a distance, over 3- to 5-cell diameters to set the boundaries of the Serrate expression domain and thus establishes conditions for subsequent juxtacrine signaling. Long-range Wingless action is also required for the asymmetric action of Serrate: Serrate does not activate veinlet (rhom) expression posteriorly because of the presence of Wingless there, 3- to 5-cell diameters from the site of wingless transcription. In this sense, Wingless modulates, at a distance, the outcome of local signaling. In neither of these activities is there evidence for concentration-dependent signaling. However, one cannot formally exclude the possibility that the specification of type 6 denticle requires low-level Wingless. Furthermore, the suggestion that Wingless is not a morphogen in the embryonic epidermis is at odds with studies of the first thoracic segment where various levels of Wingless signaling lead to the specification of distinct cuticular structures. Re-assessment of these phenotypes with early molecular markers might tell whether or not Wingless acts directly in a concentration-dependent manner in the embryonic epidermis (Alexandre, 1999).

The situation with Hedgehog is clearer since it has qualitatively distinct effects over a narrow strip of cells. It activates veinlet expression in adjoining posterior cells while its repressive effect on Serrate expression extends over three cell diameters. This suggests that, at high level, Hedgehog activates veinlet (near the source) while at both low and high levels it repress Serrate expression (further away from the source). In this sense, Hedgehog qualifies as a morphogen. Whether differential responses at different distances from the Hedgehog source reflect true concentration dependence remains to be assessed. It is noted that the repressive effect of Hedgehog on Serrate expression might take place early in development since, in wingless mutants, hedgehog expression decays around stage 10 and yet Serrate expression is still confined at the anterior. It is suggested that early Hedgehog has a repressive effect on Serrate expression that lasts at least until stage 11, when veinlet expression commences. It is therefore conceivable that the 3-cell-wide domain where Serrate is repressed at stage 11 originates by cell proliferation from a single row of cells that abut the Hedgehog source at early embryonic stages. According to this scenario, the effects of Hedgehog on Serrate and veinlet expression would both be occurring over one cell diameter. The apparent difference in range would reflect the difference in timing between these two effects and the intervening proliferation. This model is being tested by assessing the activity of a membrane-tethered form of Hedgehog (Alexandre, 1999).

To sum up, in the bald area of abdominal segments, one cell type forms in response to one signaling pathway while within denticle belts, a rich pattern of cell types arise from juxtacrine cell interactions initiated by the activation of distinct signaling pathways. Some of these pathways are controlled by the localized expression of segment polarity genes such as wingless and hedgehog, while others are regulated by downstream genes like veinlet and Serrate. Because wingless and hedgehog are expressed first, they are effectively at the top of the hierarchy and the knock-on effects of losing hedgehog or wingless function explain the 'organizer activity' of the parasegment boundary. Interestingly, the denticle Hedgehog originating from the parasegment boundaries of adjacent segments (and therefore, two parasegment boundaries) are needed to provide the signals that pattern a single denticle belt (Alexandre, 1999).

Notch signaling controls cell fate specification along the dorsoventral axis of the Drosophila gut: rho is a Notch target gene

The genetic programs that control patterning along the gut dorsoventral (DV) axis have remained largely elusive. The activation of the Notch receptor occurs in a single row of boundary cells that separates dorsal from ventral cells in the Drosophila hindgut. rhomboid, which encodes a transmembrane protein, and knirps/knirps-related, which encode nuclear steroid receptors, are Notch target genes required for the expression of crumbs, which encodes a transmembrane protein involved in organizing apical-basal polarity. Notch receptor activation depends on the expression of its ligand Delta in ventral cells, and localizing the Notch receptor to the apical domain of the boundary cells may be required for proper signaling. The analysis of gene expression mediated by a Notch response element suggests that boundary cell-specific expression can be obtained by cooperation of Suppressor of Hairless and the transcription factor Grainyhead or a related factor. These results demonstrate that Notch signaling plays a pivotal role in determining cell fates along the DV axis of the Drosophila hindgut. The finding that Notch signaling results in the expression of an apical polarity organizer, one which, in turn, may be required for apical Notch receptor localization, suggests a simple mechanism by which the specification of a single cell row might be controlled (Fusse, 2002).

While studying the role of the kni and knrl genes which act redundantly during hindgut development, it was observed that both genes are also coexpressed in the large intestine, from germ band extension stage onward in two rows of lateral cells (20 ± 1) on each side of the tube. This expression is maintained until late stage 16. In the lateral cell rows, kni and knrl are coexpressed with the rhomboid (rho) gene that encodes a transmembrane protein involved in epidermal growth factor receptor (Egfr) signaling. rho gene expression in the lateral cells appears slightly earlier than kni/knrl gene expression and is also maintained until late stage 16. At the transitions to the small intestine and the large intestine, rho is expressed in two circular expression domains. The transmembrane protein and apical polarity determinant Crumbs (Crb) becomes strongly upregulated in the lateral cell rows after germ band extension stage and displays an unusual cellular distribution. This contrasts with the dorsal and ventral cells of the hindgut, where Crb is located at the apical cell margins -- Crb is localized to the entire apical domain of the lateral cell rows, and expression is maintained until the end of embryogenesis. Similarly, Discs lost (now redefined as Drosophila Patj), another apical polarity organizer, is located to the entire apical cell surface in these cells (Fusse, 2002).

Using various cell shape and cell polarity markers, such as the septate junction markers Fas III, Neurexin IV, and Discs lost, it was determined that the cells of the lateral cell rows show a flat and long-shaped morphology and that these cells separate homogenous cell populations in the dorsal and the ventral halves of the large intestine. The cells of the lateral cell rows can thus be considered boundary cells separating dorsal from ventral cells in the large intestine. The dorsal cells, which are big and columnar, express the homeodomain protein Engrailed (En) from extended germ band stage onward until late embryogenesis. In contrast, the ventral cells, which are small and cuboidal, display expression of Delta from extended germ band stage onward until late embryogenesis. Double immunostainings reveal that En expression in the dorsal half of the large intestine is adjacent and nonoverlapping to the kni/knrl/rho expression domains in the boundary cells. Similarly, the Delta expression domain in the ventral half is adjacent to the boundary cells, although coexpression at a low level in the boundary cells cannot be excluded. In summary, dorsal cells express En; boundary cells kni/knrl, rho, crb, and ventral cells express Delta (Fusse, 2002).

To investigate the role of the genes expressed in the large intestine, lack- and gain-of-function studies were performed. In amorphic Notch and Delta mutant embryos, kni/knrl, rho, and high levels of Crb expression on the apical plate are absent in the large intestine, and the boundary cell fate is not established. In contrast, ventral cell morphologies are normal in Notch or Delta mutant embryos, and En expression and dorsal cell fates are unchanged. This indicates that Notch signaling is required to establish the boundary cells but not for dorsal or ventral cell fates. To further test this, gain-of-function experiments were performed using the UAS/Gal4 system. As driver lines, the G445.2 or the 14-3-fkhGal4 strains were used -- they mediate ubiquitous gene expression in the developing hindgut from the extended germ band stage onward until late stage 16. In order to ectopically activate the Notch signaling pathway, flies carrying the Notch intracellular domain fragment, Nicd, under the control of UAS sequences were used. Expressing Nicd ubiquitously in the hindgut results in an ectopic induction of kni and of rho. In addition, the cellular localization of the Crb protein is affected in these embryos. In dorsal and ventral cells of the large intestine of wild-type embryos, Crb is localized to the apical cell margins, whereas it is localized to the entire apical plates of the boundary cells. In the embryos, in which Nicd is ectopically expressed, Crb protein is found on the apical plates of all the hindgut cells; in addition, it is found in high concentrations in vesicles, especially on the baso/lateral sides of the cells. A similar but less intensive ectopic expression of Crb can also be induced if both Kni and Rho are coexpressed in all the hindgut cells, suggesting that crb may be a downstream effector gene of Kni/Knrl and Rho activities. This is consistent with the analysis of rho7M; Df(3L) riXT1 mutants [Df(3L) riXT1 is a deficieny encompassing the kni and knrl transcription units] in which the expression of crb in the boundary cells is strongly reduced. In summary, these results suggest that rho, kni/knrl, and Crb are target genes which are activated in response to Notch signaling in the boundary cells (Fusse, 2002).

To investigate the relationship between rho and kni/knrl in the boundary cells, the expression of the genes in the respective mutants was examined. rho expression is still present in kni mutants and Df(3L) riXT1 mutants. Similarly, kni and knrl expression are maintained in amorphic rho7M mutants or EGF receptor mutants, such as faint little ball (flb). In flbIK35 mutants, the hindgut tube is much shortened due to a reduction of the cell number. However, banded expression of both genes is found in the small intestine and the rectum along the AP axis of the hindgut; expression is also found in a few cells in the large intestine region. Ectopic expression of rho using the corresponding UAS-effector line combined with a driver line that mediates ubiquitous expression in the hindgut does not result in ectopic kni/knrl gene expression and vice versa. This points toward rho and kni/knrl being regulated independently of one another (Fusse, 2002).

To study whether En, which is expressed in the adjacent dorsal cells, contributes to the boundary cell fate, the expression of kni/knrl, rho, and crb was examined in en mutants and in en; invected double mutants (enE), since en and invected are known to act redundantly. Whereas the expression of the Notch target genes remains unchanged in en mutants, it is absent in the large intestine of en; invected double mutants. Morphological studies indicate that the dorsal and the boundary cell fates are not established in these mutants, and the large intestine seems to consist entirely of the ventral cell fates. To investigate the cause for this effect, the expression of Delta was studied in these mutants and it was found to be expressed ubiquitously in the large intestine. These data indicate that a boundary between Delta expressing and nonexpressing cells is required for Notch receptor activation. Ectopic expression of En in the large intestine using the 14-3 fkh driver and UAS-En effector lines results in a repression of kni/knrl and rho gene expression. This indicates that En bears the potential to act as a negative regulator of Notch target genes. Upon ectopic activation of Notch signaling in the entire hindgut by expressing Nicd, En is repressed on the dorsal side of the large intestine, thus allowing ectopic activation of Notch target genes (Fusse, 2002).

These results demonstrate that Notch signaling induces the expression of the rho and kni/knrl genes and that both components are required, in turn, for the expression of Crb. It has been suggested recently that Su(H) functions as a core of a molecular switch by which the transcription of Notch target genes is regulated. In the absence of Notch signaling, Su(H) functions as a repressor, and, in the presence of Notch signaling, Su(H) can cooperate synergistically with other transcriptional activators to induce transcription of target genes. The finding that boundary cell-specific reporter gene expression can be induced in the hindgut by using a model Notch response element [composed of binding sites for Su(H) and the widely expressed activator Grainyhead] suggests the possibility that the localized activation of the rho and kni/knrl genes could rely on the same factors and the same molecular switch mechanism that has recently been proposed for this element and for Notch-dependent atonal and single minded expression. In evolutionary terms, the gut is most likely one of the most ancient organs that evolved in multicellular organisms. Consistently, the morphological processes involved in the development of the gastrointestinal tract of animals are highly similar. It remains to be shown whether or not the evolutionarily conserved regulators of the Notch signaling cascade also determine dorsoventral aspects of gut development in other animals, including vertebrates (Fusse, 2002).

abdominal A specifies one cell type in Drosophila by regulating one principal target gene: Regulation of rho expression

The Hox/homeotic genes encode transcription factors that generate segmental diversity during Drosophila development. At the level of the whole animal, they are believed to carry out this role by regulating a large number of downstream genes. This study addresses the unresolved issue of how many Hox target genes are sufficient to define the identity of a single cell. Focus was placed on the larval oenocyte, which is restricted to the abdomen and induced in response to a non-cell autonomous, transient and highly selective input from abdominal A (abdA). Hox mutant rescue assays were used to demonstrate that this function of abdA can be reconstituted by providing Rhomboid (Rho), a processing factor for the EGF receptor ligand, secreted Spitz. Thus, in order to make an oenocyte, abdA regulates just one principal target, rho, that acts at the top of a complex hierarchy of cell-differentiation genes. These studies strongly suggest that, in at least some contexts, Hox genes directly control only a few functional targets within each nucleus. This raises the possibility that much of the overall Hox downstream complexity results from cascades of indirect regulation and cell-to-cell heterogeneity (Brodu, 2002).

Potential abdA targets were sought from among the genes known to play a role in the specification or function of C1. This particular sensory organ precursor produces a type of stretch receptor, the chordotonal organ, that is defined by the proneural gene atonal (ato). ato is also required for oenocyte formation but it is similarly expressed in thoracic and abdominal C1, is not regulated by abdA and is downregulated prior to oenocyte induction. rho, a gene downstream of ato and rate-limiting for the production of sSpi by cleavage from an inactive membrane-bound precursor (mSpi) in the Golgi apparatus, was examined. Like ato, rho is also required for oenocyte formation. Rho protein is first expressed in C1 at stage 10, after it has delaminated from the dorsal ectoderm. As with Ato at this stage, early Rho is present at similar levels in thoracic and abdominal C1 precursors and is not under abdA control. During stage 11, however, thoracic Rho becomes extinguished while abdominal Rho persists at a similar level in the C1 lineage. Unlike the early expression, this late phase correlates with the time of oenocyte induction and is missing in abdA mutants. Furthermore, driving AbdA in the C1 lineage during stage 11, either in the thorax of a wild-type embryo, or in the abdomen of an abdA mutant, is sufficient to prolong Rho expression. Together, these results indicate that the maintenance but not the establishment of Rho expression is under abdA control. Analysis of a rho-lacZ line, expressed at stage 11 but not stage 10, suggests that this late regulation is at the transcriptional level and is mediated by a different enhancer than that controlling the early phase of expression (Brodu, 2002).

Next, it was asked whether the rather simple Rho timing difference between the thorax and the abdomen is responsible for deciding whether a segment is going to form oenocytes. en-GAL4 or ato-GAL4 were used to extend the time-window of Rho expression in the thorax. Remarkably, using either driver results in the formation of bona fide oenocytes, albeit that they are frequently unclustered and dorsally misplaced. The sufficiency of rho in the absence of AbdA can be clearly demonstrated since ato-GAL4 driven expression of Rho rescues oenocyte formation in abdA mutants. Hence, prolonging the expression of Rho in the C1 lineage is all that is needed to reconstitute the oenocyte identity function of abdA. Next, ato-GAL4 was used to drive AbdA or Rho expression in Scr;Antp double homozygotes. Since ectopic oenocytes are formed in both cases in the mutant T1 segment, representing the ground state, any redundant requirement in the responding ectoderm arising from functional equivalence of Hox proteins can be ruled out (Brodu, 2002).

The above experiments do not reveal whether C1 also produces some other oenocyte signal that is normally present in both thorax and abdomen. Addressing this issue, en-GAL4 was used to express Rho in a genetic background lacking ato, and therefore missing a functional C1 cell. In this mutant context, oenocytes can still be induced, indicating that the only role that C1 plays during oenocyte specification is to express Rho and thus provide a source of sSpi signal (Brodu, 2002).

Maintenance of Rho expression by abdA is predicted to extend the period of Spi secretion, so that the abdominal C1 lineage signals for longer than its thoracic homolog. To test directly whether prolonging active ligand production could induce oenocytes, ato-GAL4 was used to drive a constitutively active form of sSpi, in the C1 lineage after stage 10. This resulted in ectopic oenocytes in the thorax and, more importantly, rescued oenocyte formation in abdA mutants. Providing sSpi prematurely, from stage 9 onwards using en-GAL4, also produces thoracic and rescued abdominal oenocytes but the onset of induction remains restricted to the normal time window during stage 11. Together, these results demonstrate that the oenocyte specification function of abdA can be rescued by adding back either Rho or sSpi in C1 during the period of ectodermal competence. Given that the oenocyte role of abdA is synonymous with prolonging Rho and thus sSpi synthesis in C1, then activating the Egfr in the dorsal ectoderm at the appropriate time would be expected to have the same effect. Consistent with this prediction, expressing constitutively active Egfr (EgfrACT) under the control of sal-GAL4 is sufficient to trigger oenocyte formation in abdA mutants, completely rescuing their number, position and clustering (Brodu, 2002).

Oenocyte formation is under the positive control of AbdA and its co-factor Exd. The temporally restricted pulse of AbdA expression in C1 reflects a transient function in prolonging the oenocyte-inducing signal during stage 11. This type of hit-and-run Hox function appears to be widespread and has previously been observed for other ectodermal derivatives. The misexpression experiments clearly indicate that the oenocyte-promoting role of AbdA is highly selective and can not be substituted for by Ubx. This is explained in molecular terms, since only AbdA is capable of maintaining the transcription of rho in the C1 lineage. Such selectivity contrasts with the equivalent biological activities of Ubx and AbdA proteins in promoting haltere formation. In this regard, it is noted that exd is required to make an oenocyte but not a haltere and therefore may allow these two Hox proteins to discriminate between different targets (Brodu, 2002).

In the absence of any Hox input, oenocytes are completely missing and therefore are not an overt part of the ground state. At first sight, it might seem that for cell types that have no morphological representation in the ground state, such as oenocytes, Hox genes must necessarily play a classic instructive role in defining the appropriate pathway of differentiation. However, as will now be argued, this is not the only way that Hox genes can direct the formation of segment-specific cell types. Two lines of evidence suggest that the sSpi signal from C1 is permissive in the sense that it does not itself contain any oenocyte specificity information: (1) providing ectopic sSpi signal outside of a restricted dorsal zone around C1 fails to induce oenocytes; (2) the degree of sSpi signaling influences the number of induced cells rather than their identity. In contrast, it has been demonstrated that all of the cell-type specificity information is encoded in the dorsal ectoderm as an oenocyte prepattern. One crucial component of this prepattern is encoded by sal. The Sal zinc-finger transcription factor acts to prime the Egfr response in favour of the oenocyte fate. In its absence, there is a fate switch and sSpi signaling now induces secondary chordotonal organs. Thus, it has been shown that oenocyte specificity is provided by the sal-dependent prepattern and not by the sSpi-inducing signal (Brodu, 2002).

The segmental restriction of oenocyte induction has been analysed and evidence is provided supporting a model where there is no Hox input into the prepattern but the timing of the sSpi-inducing signal is controlled by abdA. Together with the previous finding that sSpi signaling is permissive, it is now concluded that abdA does not directly specify the oenocyte identity, rather it determines which segments will form oenocytes. This involves modifying the signaling properties of C1, a serially reiterated cell type that is part of the ground state. In turn, this provides a permissive trigger that uncovers a cryptic oenocyte identity also present in the ground state. Hence ato and sal, two of the genes that contribute to the ground state, are essential for specifying the C1 cell type and the complete oenocyte prepattern, respectively. Another important feature of this model is that the dorsal ectoderm is not competent for oenocyte induction until stage 11. This implies that if competence were to be acquired earlier, when C1 expresses Rho in both the thorax and abdomen, then oenocytes would be produced in all trunk segments independent of Hox genes (Brodu, 2002).

Cholesterol modification of Hedgehog is required for trafficking and movement, revealing an asymmetric cellular response to Hedgehog: Regulation of rho expression

Hedgehog family members are secreted proteins involved in numerous patterning mechanisms. Different posttranslational modifications have been shown to modulate Hedgehog biological activity. The role of these modifications in regulating subcellular localization of Hedgehog has been investigated in the Drosophila embryonic epithelium. Cholesterol modification of Hedgehog is responsible for Hedgehog assembly in large punctate structures and apical sorting through the activity of the sterol-sensing domain-containing Dispatched protein. Movement of these specialized structures through the cellular field is contingent upon the activity of proteoglycans synthesized by the heparan sulfate polymerase Tout-Velu. Finally, the Hedgehog large punctate structures are necessary only for a subset of Hedgehog target genes across the parasegmental boundary, suggesting that presentation of Hedgehog from different membrane compartments is responsible for Hedgehog's functional diversity in epithelial cells (Gallet, 2003).

The repeated pattern of the Drosophila larval ectoderm (which secretes cuticle) has been used to follow Hh activity. Each abdominal segment is composed of two types of cuticle: the naked (or smooth) cuticle and the denticle belts, subdivided into six rows of denticles, easily identifiable by their orientation and shape. This cuticle pattern is under the control of several signaling pathways that are indirectly regulated by Hh. Engrailed (En) controls hh expression in the two rows of cells that define the posterior compartment of the segment. Across the parasegmental boundary (in cells anterior to the En/Hh domain), Hh maintains wingless (wg) transcription in one row of cells. The Wg signal then controls the specification of the naked cuticle. Posterior to the En/Hh domain, Hh initiates rhomboid (rho) transcription in one to two rows of cells. rho activation induces EGF signaling, allowing differentiation of denticles 1-4. Finally, Hh and Wg are required for serrate (ser) repression and restrict its expression in three rows of cells posterior to the rho-expressing cells. Ser initiates a third row of rho expression in adjacent cells. The Hh receptor Patched (Ptc) is also transcriptionally upregulated by the Hh pathway in cells on both sides of the En/Hh domain (Gallet, 2003).

Loss of hh results in loss of both naked cuticle and denticle diversity. This cuticle phenotype correlates with Hh target gene expression: loss of wg, extension of the ser expression domain (which now covers most of the segment) and absence of ptc upregulation. rho expression is strongly reduced, though some remains under the control of Ser. Conversely, ubiquitous expression of full-length hh (HhFL) in the ectoderm with the GAL4-UAS system induces an expansion over four to five cells of both wg and rho expression in the anterior and posterior directions, respectively, while ser expression is completely repressed. Accordingly, the denticle belts of these embryos contain several rows of type 2 denticles, reflecting a uniform level of rho expression in response to a uniform level of Hh. Thus, wg, rho, ser, and ptc expressions reflect direct Hh activity in cells anterior and posterior to en/hh-expressing cells (Gallet, 2003).

Two endogenous Hh isoforms are present in vivo: one bearing both posttranslational lipid modifications and another modified only by a cholesterol adduct. To address the role of these different modifications in Hh signaling, the biological activity of different Hh constructs that do not undergo all modifications was assessed (Gallet, 2003).

It is hypothesized that the differences observed could be accounted for by differential activation mechanisms. These results outline the important role of the Hh cholesterol modification in stimulating the anterior target genes wg and ptc across the parasegmental boundary and, subsequently, naked cuticle differentiation, while cholesterol appears dispensable for posterior induction of ptc and rho and, thus, denticle diversity. Because some wg expression can still be activated by Hh-N, the presence of cholesterol modification on Hh might not be the only requirement for anterior target gene regulation. Hh-N-CD2 and Hh-N-GPI activities suggest that the differences observed could be a consequence of Hh differential sorting in the producing cells and/or access and presentation to the target cell surface (Gallet, 2003).

Differential activation of wg and ptc in anterior cells and of rho and ptc in posterior cells is related to the membrane localization of Hh. Cholesterol-dependent LPS formation and apical targeting are shown to be necessary for proper anterior wg activation but dispensable for rho expression in posterior cells. Conversely, basolateral targeting of Hh in cells producing Hh-N-CD2 and Hh-N-GPI is sufficient to activate the posterior rho expression, independent of the presence of cholesterol (Gallet, 2003).

Interestingly, wg is expressed in adjacent cells located just anterior to the Hh-sending cells. Hence, long-range diffusion of Hh should not be required for wg activation. However, in the absence of Ttv function, Hh-Np LPSs are blocked apically in producing cells, and wg is not activated. Ttv-dependent heparan sulfate proteoglycans are required for long-range Hh-Np movement in the wing disc. Thus, these results suggest that, in the embryonic ectoderm, two different mechanisms of Hh pathway activation are present. wg activation requires all the events previously associated with long-range Hh target gene activation and thus depends on Hh secretion and transport mechanisms. However, rho does not require secretion of Hh and can be activated in a cell-cell contact-dependent manner, like a short-range target. This difference could be due to differential accessibility of Hh to anterior versus posterior cells caused by the presence of the parasegmental boundary between en and wg cells. Indeed, when Ttv is expressed exclusively in cells anterior to En cells, both wg- and rho-dependent cell differentiation are rescued. This indicates that a differential transport and/or presentation of Hh-Np could be responsible for the asymmetric cellular response to Hh (Gallet, 2003).

How then is rho activated in cells posterior to Hh-producing cells? rho expression could depend on cell-cell contact activation with or without internalization of Hh. Although no detectable Hh in Hh-N-CD2 neighboring cells was observed, the possibility cannot be excluded that rho activation might depend on Hh internalization. It is worth mentioning that an Shh-CD4 transmembrane fusion protein has been shown to be internalized in adjacent cells through Ptc-1 activity in mammalian tissue culture cells and can induce formation of the most posterior digit of the chick limb. Moreover, expression of Hh-Np in disp mutant embryos that are defective in apical sorting induces rho expression in several rows of cells. In these embryos small dots of Hh-Np are seen outside the producing cells, confirming a possible internalization of Hh in posterior receiving cells through basolateral membrane interactions. This internalization could propagate at long range, since rho and ptc are activated in six to seven rows of cells when non-cholesterol-modified Hh-N is expressed in disp mutant embryos (Gallet, 2003).

In summary, these data suggests that some Hh/Shh targets can be activated through Hh trafficking in LPSs followed by apical secretion, whereas other targets might be activated by basolaterally targeted Hh. Hence, it is hypothesized that presentation of Hh from different cellular membrane compartments allows the receiving cells to differentially respond to the Hh input. This provides an interesting new paradigm regarding the mode of action of morphogens in all metazoans (Gallet, 2003).

Hox-controlled reorganisation of intrasegmental patterning cues underlies Drosophila posterior spiracle organogenesis: Egfr pathway provides specific input for posterior spiracle morphogenesis

Hox proteins provide axial positional information and control segment morphology in development and evolution. Yet how they specify morphological traits that confer segment identity and how axial positional information interferes with intrasegmental patterning cues during organogenesis remains poorly understood. This study investigates the control of Drosophila posterior spiracle morphogenesis, a segment-specific structure that forms under Abdominal-B (AbdB) Hox control in the eighth abdominal segment (A8). The Hedgehog (Hh), Wingless (Wg) and Epidermal growth factor receptor (Egfr) pathways provide specific inputs for posterior spiracle morphogenesis and act in a genetic network made of multiple and rapidly evolving Hox/signalling interplays. A major function of AbdB during posterior spiracle organogenesis is to reset A8 intrasegmental patterning cues, first by reshaping wg and rhomboid expression patterns, then by reallocating the Hh signal and later by initiating de novo expression of the posterior compartment gene engrailed in anterior compartment cells. These changes in expression patterns confer axial specificity to otherwise reiteratively used segmental patterning cues, linking intrasegmental polarity and acquisition of segment identity (Merabet, 2005).

In the dorsal ectoderm of stage 10 embryos, hh and wg follow the same striped expression patterns in A8 as in other abdominal segments. rho expression, which marks cells secreting an active form of the Egf ligand, occurs in all primordia of tracheal pits, in A8 as in more anterior segments (Merabet, 2005).

Specification of posterior spiracle primordia occurs at early stage 11. The primordia can then be recognised by Cut expression in spiracular chamber cells and by Sal, the homogenous expression of which in A8 becomes restricted dorsally to stigmatophore cells (forming the external structure of the posterior spiracle) that form a crescent surrounding Cut-positive cells. From mid-stage 11, wg and rho adopt in the dorsal ectoderm expression patterns specific to A8, with wg transcribed in two cells only and rho in a second cell cluster, dorsal and posterior to the tracheal placode. To localise wg- and rho-expressing cells with regard to stigmatophore and spiracular chamber cells, co-labelling experiments for wg or rho transcripts and for Cut or Sal proteins were performed: the two wg cells lie between Cut- and Sal-positive cells; the second cell cluster expressing rho in A8 also expresses Cut but not Sal. This cluster is likely to produce the Egf ligand required for posterior spiracle development, since mutations that alleviate rho expression in the tracheal placodes do not abolish spiracles formation. At mid-stage 11, the hh pattern in A8, along a stripe lying posterior and adjacent to the spiracular chamber and overlapping stigmatophore presumptive cells, resembles expression in other abdominal segments. Analyses at later stages indicate that the relationships between posterior spiracle cells and hh, wg and rho patterns are maintained (Merabet, 2005).

A8-specific modulation of rho and wg patterns at mid-stage 11 suggests a regulation by AbdB. In AbdB mutants, rho expression in the spiracle-specific cell cluster is lost, and wg transcription does not evolve towards an A8-specific pattern. In embryos expressing AbdB ubiquitously, ectopic posterior spiracle formation in the trunk can be identified as ectopic sites of Cut accumulation. In such embryos, rho and wg are induced in trunk segments following patterns that resemble their expression in A8: rho in a cluster that overlaps the Cut domain, and wg in few cells abutting ectopic Cut-positive cells. These transcriptional responses to loss and gain of function of AbdB indicate that the Hox protein controls the A8-specific expression patterns of wg and rho. The lines gene (lin), which is known to be required for Cut and Sal activation by AbdB, also controls wg and rho patterns respecification (Merabet, 2005).

In contrast to wg and rho, hh does not adopt an A8-specific expression pattern at mid-stage 11. At that stage, hh expression pattern is not affected upon AbdB mutation. The hh stripe in A8 lies posterior and adjacent to spiracular chamber cells and overlaps stigmatophore cells, suggesting that Hh signalling may participate in the regulation of rho and wg transcription by AbdB. In support of this, it was found that the AbdB-dependent aspects of rho and wg transcription patterns are missing in hh mutant embryos. Thus, inputs from both Hh and AbdB are required to remodel Wg and Egfr signalling in A8 (Merabet, 2005).

The dependence of wg and rho A8 expression patterns on Hh, and the loss of ems expression in wg and rho but not in hh mutants, suggest that transcription of ems requires Wg and Egfr signalling prior to wg and rho pattern respecification by AbdB and Hh. To explore this point further, the time course of ems, wg and rho expression was comparatively analyzed. Embryos bearing an ems-lacZ construct stained for ß-Gal and for wg or rho transcripts show that ems expression precedes wg pattern respecification, and occurs at the same time as rho acquires an A8-specific pattern. Importantly, A8-specific rho clusters were never observed before the onset of ems expression. Thus, ems transcription starts before wg and at the same time as rho pattern respecification, supporting that signalling by Wg and Egfr is required prior to mid-stage 11. These observations also indicate that respecification of the wg pattern occurs slightly later than that of rho, which could not been concluded from changes in embryo morphology (Merabet, 2005).

To determine whether signalling by Wg and Egfr from local sources is important for posterior spiracle organogenesis, the production of Wg and SpiS (the mature form of Spi) ligands was forced from domains broader than normal in A8 dorsal ectoderm. This was performed after posterior spiracle specification, using the ems-Gal4 and sal-Gal4 drivers. Ectopic signalling results in abnormally shaped posterior spiracles: stigmatophores are reduced in size and filzkörpers do not elongate properly. Ectopic signalling from all presumptive stigmatophore cells results in stronger defects than those produced when ectopic signals emanate from all spiracular chamber cells. This can be correlated to the fact that sal-Gal4 drives expression in a pattern that more strongly diverges from the wild-type situation than ems-Gal4 does. Thus, restricted delivery of Wg and SpiS signals is required for accurate posterior spiracle organogenesis (Merabet, 2005).

It was next asked whether, downstream of Hh, the Wg and Egfr pathways provide separate inputs for posterior spiracle organogenesis. Two sets of experiments were conducted and it was found that: (1) in embryos respectively mutant for Egfr or wg, wg and rho acquire A8-specific patterns; (2) epistasis experiments performed by forcing in spiracular or stigmatophores cells the activity of one pathway while inhibiting the other indicate that loss of one pathway could not be rescued by the other. Thus, Egfr and Wg pathways do not act as hierarchically organised modules, but provide independent inputs for posterior spiracle organogenesis (Merabet, 2005).

The expression of the posterior compartment selector gene engrailed (en) until stage 12 follows a striped pattern identical in all trunk segments. Later on, En adopts a pattern that is specific to A8: it is no longer detected in the ventral part of the segment; dorsally, the En stripe has turned to a circle of cells that surround the future posterior spiracle opening and express the stigmatophore marker Sal. The transition from a striped to a circular pattern depends on AbdB. This transition could result either from a migration of en posterior cells towards the anterior, or from transcriptional initiation in cells that were not expressing en before stage 12, and that can therefore be defined as anterior compartment cells (Merabet, 2005).

To distinguish between the two possibilities, en-Gal4/UAS-lacZ embryos were simultaneously stained with anti ß-Gal and anti-En antibodies. If circle formation results from cell migration, one would expect ß-Gal and En to be simultaneously detected in all cells of the circle since the two proteins are already co-expressed in the posterior compartment stripe earlier on. Conversely, if the circle results from de novo expression, one would expect anterior cells in the circle to express En before ß-Gal, since ß-Gal production requires two rounds of transcription/translation compared with one for En. It was found that cells from the anterior part of the circle express En but not ß-Gal in stage 13 embryos, which demonstrates that de novo expression of En occurs in anterior compartment cells. Further supporting En expression in anterior compartment cells, it was found that precursors of anterior spiracle hairs that do not express En at stage 12 do so at stage 13. Engrailed function in A8 is essential for posterior spiracle development, since stigmatophores do not form in en mutants, and are restored if En is provided in stigmatophore cells (Merabet, 2005).

It was also found that although identical in all abdominal segments at stage 11, hh transcription adopts an A8-specific pattern from stage 12 onwards: transcripts are then localised only at the anterior border of the En stripe. This expression of hh is lost in AbdB mutants and still occurs in en mutant. The uncoupling of hh transcription from En activity in the dorsal A8 ectoderm correlates with the distinct phenotypes seen for en mutants, which do differentiate filzkörper like structures, and for hh mutants, which do not (Merabet, 2005).

Data in this paper allow the distinguishing of four phases in functional interactions between AbdB and signalling by Wg, Hh and Egfr during posterior spiracle formation. The first phase corresponds to the specification of presumptive territories of the organ. The signalling activities are not involved in this AbdB-dependent process, since they are not required for the induction of the earliest markers of spiracular chamber and stigmatophore cells, Cut and Sal, in the dorsal ectoderm of A8. The second phase, which immediately follows primordia specification, concerns the regulation of AbdB target genes activated slightly later. Inputs from the Hox protein and the Wg and Egfr pathways are then simultaneously needed, as seen for transcriptional initiation of the ems downstream target. This function of Wg and Egfr signalling precedes and does not require the reallocation of signalling sources in A8-specific patterns; impairing A8-specific expression of wg and rho by loss of hh signalling does not affect ems expression. Within the third phase, AbdB and Hh activities converge to reset wg and rho expression patterns. The three phases take place in a narrow time window, less than 1 hour during stage 11, and could only be distinguished by studying the functional requirements of Wg, Hh and Egfr for transcriptional regulation in the posterior spiracle (Merabet, 2005).

The fourth phase is referred to as an organogenetic phase. Data obtained using DN variants to inhibit the pathways in cells already committed to stigmatophore or filzkörper fates, indicate that Wg, Egfr and Hh pathways are required for organ formation after specification and early patterning of the primordia. Their roles are then to maintain the AbdB downstream targets' expression in posterior spiracle cells as development proceeds, as shown for Cut and Sal at stage 13 (Merabet, 2005).

A salient feature of AbdB function during posterior spiracle development is to relocate Wg and Egfr signalling sources in the dorsal ectoderm at mid-stage 11. wg and rho then adopt expression patterns that differ from expressions in other abdominal segments, conferring axial properties unique to A8 to otherwise segmentally reiterated patterning cues. Resetting Wg and Egfr signalling sources into restricted territories is of functional importance for organogenesis, as revealed by the morphological defects that result from the delivery of Wg or SpiS signals in all spiracular chamber or stigmatophore cells after the specification phase. During stage 12, AbdB also relocates the Hh signalling source by inducing En-independent expression of hh in the dorsal ectoderm. Thus, later than Wg and Egfr signalling, the Hh signal also acquires properties unique to A8. In generating this pattern, AbdB plays a fundamental role in uncoupling hh transcription from En activity, providing a context that prevents anterior compartment En-positive cells to turn on hh transcription, and that allows hh expression in the absence of En in other cells. Slightly later, at stage 13, AbdB modifies the expression of the posterior selector gene en, initiating de novo transcription in anterior compartment cells. In these cells, En fulfils different regulatory functions than in posterior cells, as discussed above for hh regulation. Changes in En expression and function can be interpreted as a requisite to loosen AP polarity in A8 and gain circular coordinates required for stigmatophore formation (Merabet, 2005).

Osa, a subunit of the BAP chromatin-remodelling complex, participates in the regulation of gene expression in response to EGFR signalling in the Drosophila wing

Gene expression is regulated in part by protein complexes containing ATP-dependent chromatin-remodelling factors of the SWI/SNF family. In Drosophila there is only one SWI/SNF protein, named Brahma, which forms the catalytic subunit of two complexes composed of different proteins. The protein Osa defines the Bramha associated protein (BAP) complex, and the proteins Polybromo and Bap170 are only present in the complex named PBAP. This work analysed the functional requirements of Osa during Drosophila wing development, and found that osa is needed for cell growth and survival in the wing imaginal disc, and for the correct patterning of sensory organs, veins and the wing margin. Other members of the BAP complex, such as Snr1, Bap55, Mor (Moira) and Brahma, also share these functions of Osa. Focus was placed on the requirement of Osa during the formation of the wing veins. Genetic interactions between osa alleles and mutations affecting the activity of the EGFR pathway suggest that one aspect of Osa is intimately related to the response to EGFR activity. Thus, loss of osa and EGFR signalling results in similar wing vein phenotypes, and osa alleles enhance the loss of veins caused by reduced EGFR activity. In addition, Osa is required for the expression of several targets of EGFR signalling, such as Delta, rhomboid and argos. It is suggested that one role of Osa and Brm in the wing is to establish a chromatin environment in the regulatory regions of EGFR target genes, making them available for both activators and repressors and facilitating transcription in response to EGFR signalling (Terriente-Félix, 2009).

Chromatin structure is critical to modulate gene expression during development, and is affected by a variety of alterations such as histone modification, DNA methylation and changes in conformation. Proteins related to Drosophila Brm, such as yeast SNF2 modify chromatin in an ATP-dependent manner, causing repositioning of nucleosomes along the DNA and re-distribution of histone proteins between nucleosomes. The SWI/SNF complexes are conserved in all eukaryotes, and display specific interactions with distinct transcription factors to regulate different subsets of genes. There are several examples where sequence-specific transcription factors interact specifically with SWI/SNF complexes. For example, the ATPase BRG1 binds Zn-finger proteins and hBRM interacts specifically with CBF-1/Su(H), which recruits hBRM to Notch target promoters such as those of HES1 and HES5 (Terriente-Félix, 2009).

A key aspect in the analysis of Brm function is the identification of targets accounting for the functions of the complex. A necessary step in this analysis is the description of its functional requirements using genetic approaches; which helps to identify the specific processes affected by loss of BAP function. The current data indicate that Osa is required during wing disc development for cell viability, cell proliferation, and for the formation of wing veins and the wing margin. Interestingly, increased expression of Osa in the wing also causes phenotypes related to wing growth and patterning, such as reduced wing size, ectopic sensory organs and hairs and the formation of extra vein tissue in most interveins. This analysis focused mostly on Osa, and this raises the question of whether its requirement reflects the function of the BAP complex. This is the most likely scenario, because the preliminary analysis of other BAP members, such as Snr1, Bap55, Mor and Brm uncovers similar phenotypes in the wing. Thus, lowering Snr1, Bap55 or Mor levels reduces wing size, disrupts the wing epithelium and causes the differentiation of ectopic sensory organs and hairs. These wings also display loss of veins, and in general the overall phenotypes are similar to those of loss of Osa. The phenotype of iRNA expression directed against brm is much milder, perhaps due to a lower efficiency of this construct, but still these wings show a loss of veins phenotype. The reduction of Bap170, a member of the PBAP complex, causes the formation of ectopic veins, which is the opposite phenotype to loss of function in osa and in other members that are present in both the BAP and PBAP complexes. Thus, although Brm is the catalytic subunit in both BAP and PBAP, these complexes could act in opposite manners on the same target genes at least during wing vein formation (Terriente-Félix, 2009).

Some Osa requirements can be explained by modifications in the transcriptional response to the activity of the Wg signalling pathway and by effects on wg expression. The function of Wg is required for the formation of the wing margin, including the development of sensory organs and veins along the anterior wing margin. In the absence of Wg signalling the wing margin does not form, and when Wg signalling is inappropriately activated ectopic sensory organs and hairs differentiate throughout the wing blade. In addition to affecting the response to Wg signalling, Osa is also required for the expression of wg along the dorso-ventral boundary. This requirement might be related to Notch signalling in these cells, and explains why the remnants of wing tissue formed in osa mutant wings do not form the wing margin or ectopic sensory organs (Terriente-Félix, 2009).

This study focused on the characterisation of Osa during the formation of the longitudinal wing veins. This process is independent of Wg signalling, and requires the activities of the Notch, Dpp and EGFR signalling pathways. Osa is needed for the expression of bs in the interveins, because bs is not expressed in cells mutant for osa. The regulation of bs expression involves the activity of Ash2 and the function of the Hh and Dpp pathways. It is suggested that Osa participates in the activation of bs facilitating the availability of its regulatory regions to these activators. This aspect of Osa function does not explain the phenotype of loss of veins characteristic of osa mutant cells, because the loss of Bs expression is normally associated with the differentiation of ectopic veins. The only context where bs mutant cells differentiate as interveins is when the activity of the EGFR pathway is reduced. Therefore, it is suggested that loss of bs expression is accompanied in osa mutant cells by a failure in the response to EGFR activity, leading to the differentiation of intervein tissue. Interestingly, the expression of bs is also severely reduced when Osa is present at higher than normal levels, and in this case loss of Bs is accompanied, as expected, by the formation of ectopic veins. The effects of increased Osa on bs expression can also be explained if Osa facilitates EGFR activity, because this pathway mediates the repression of bs in the proveins. In both cases, the common aspect mediated by Osa might be to regulate bs expression in collaboration with its transcriptional activators and repressors (Terriente-Félix, 2009).

Because the failure of osa mutant cells to differentiate the veins is not due to changes in bs expression, nor to changes in the expression of provein genes such as kni and caup, the search for Osa candidate targets was narrowed to the EGFR pathway. Several results suggest a close relationship between Osa and EGFR signalling in the wing. First, the phenotypes of changing osa expression in the veins are very similar to those resulting from the same manipulation in EGFR activity. Thus, a reduction in any core component of the EGFR pathway eliminates the veins, whereas the increase in EGFR signalling activity causes the formation of extra veins in intervein territories. Second, genetic interactions were observed between osa and several components of the EGFR pathway compatible with a function of Osa promoting EGFR activity in the veins. Finally, the extra veins caused by excess of Osa are suppressed when the activity of EGFR is reduced, indicating that Osa cannot substitute for EGFR activity. The changes in vein and intervein expression patterns are already detected in the wing disc, before other signalling pathways, such as Dpp, act to promote vein formation. Taken together, these observations suggest that Osa facilitates the response to EGFR activity in the wing disc, but cannot promote the transcription of EGFR targets in the absence of EGFR signalling (Terriente-Félix, 2009).

The changes in the expression of EGFR target genes observed in osa mutant cells or in osa gain-of-function experiments are compatible with a direct function of Osa/BAP is the transcriptional regulation of EGFR targets such as Dl, rho and aos. How Osa and the BAP complex are targeted to specific genomic regions is not entirely clear, although it is likely that sequence-specific transcription factors are involved in this process. Transcription in response to EGFR signalling is mediated by proteins belonging to the ETS family, such as Pointed-P2, Pointed-P1 and Yan in Drosophila. However, these genes are not required during wing vein formation, suggesting that other ETS proteins or uncharacterised transcription factors bring about interactions between the regulatory regions of EGFR target genes and the BAP complex (Terriente-Félix, 2009).

It is unlikely that Osa participates in any step of the EGFR pathway previous to the transcription of its target genes. It was noticed, however, that the expression of dP-ERK, a direct read-out of the pathway activity, is also affected in osa mutant cells. Thus, these cells frequently fail to express normal levels of dP-ERK, a result indicating that EGFR activity is reduced. The most likely explanation for this observation is that, in the wing, the EGFR pathway is engaged in a positive feedback loop mediated by the activation of rho expression, which maintains EGFR activity in cells where it has already been activated. Thus, loss of osa leads to a failure to express rho and subsequently to a reduction in the activity of the pathway detected as a loss of dP-ERK expression. There is one experimental situation in which Osa function appears to be dispensable for the expression of EGFR target genes. Thus, when a constitutive active form of Ras, RasV12, is driven in the wing, the augmented expression of Dl and aos, and the accumulation of dP-ERK are not affected by a reduction in Osa levels. It is possible that in this situation of strong and constitutive activity of the pathway, the possible modifications to chromatin structure brought about by Osa/BAP on EGFR target genes are not necessary, perhaps because at this level of EGFR activation the transcriptional repressors antagonising EGFR target gene transcription, such as Cic and Gro, are inactivated by the pathway, and this might make dispensable the function of Osa (Terriente-Félix, 2009).

It is not entirely clear to what extent the link observed between BAP function and EGFR signalling during wing disc development is conserved in other developmental systems and in other organisms. Some phenotypes of osa and brm alleles described in the eye disc, such as the loss of photoreceptor cells, are also observed upon a reduction in EGFR activity. Similarly, the loss of distal growth in the legs is also characteristic of reduced EGFR activity. These data are indicative of a general requirement for Osa in the expression of EGFR target genes at least in imaginal discs. The genetic approach that was used identifies transcription downstream of EGFR signalling as a relevant in vivo function of BAP complexes. Subsequent biochemical analysis should determine whether the functional interactions observed are mediated by direct binding of BAP to the regulatory regions of bs and other EGFR target genes (Terriente-Félix, 2009).

Targets of Activity

Rhomboid and Ras activities prevent net transcription. Ectopic expression of rho under the control of a heat-inducible promoter during late third instar or early pupal development causes broadening of veins and ectopic vein formation, whereas absence or reduction of rho activity, or impaired Egfr-dependent signaling, generates the complementary phenotype, i.e. loss of vein structures. To test if the development of ectopic veins is linked to the repression of net, net transcription was examined in wing discs after ectopic activation of UAS-rho by MS1096. Indeed, in such discs, which express rho strongly in almost the entire disc (the pattern is identical to that of net transcripts), no net transcripts are detectable, which demonstrates that ectopic rho expression represses net transcription. It appears that in these wing discs all intervein regions develop as veins, since the resulting adult wings have a tube-like appearance and consist entirely of vein-like tissue. This phenotype is much stronger than that of net null mutants. Ectopic Rho protein suppresses intervein fate in regions where such a fate is independent of net expression. Hence, it is assumed that in these regions rho represses intervein-promoting and vein-suppression genes that are different from net, and thus activates vein-promoting genes. These genes may show partial redundancy with net functions in regions in which their expression overlaps that of net (Brentrup, 2000).

To investigate further whether rho mediates its repression of net through activated components of the Egfr signaling pathway, constitutively active Ras protein was expressed in MS1096/+;UAS-Dras1V12 /+ wing discs. Constitutive Ras activity produces overgrown wing discs that fail to express net. Although the adult wing phenotype could not be observed in these flies because they die as early pupae, ectopic activation of the Ras/mitogen-activated protein kinase (MAPK) signaling pathway in wing discs has been shown to give rise to ectopic veins. These results illustrate that ectopic expression of rho or activated components of the Egfr signaling pathway represses the transcription of net and presumably of additional vein-suppression genes, and suggest that the repression of these genes is a prerequisite for vein formation (Brentrup, 2000).

The stereotyped pattern of Drosophila wing veins is determined by the action of two morphogens, Hedgehog (Hh) and Decapentaplegic (Dpp), which act sequentially to organize growth and patterning along the anterior-posterior axis of the wing primordium. An important unresolved question is how positional information established by these morphogen gradients is translated into localized development of morphological structures such as wing veins in precise locations. In the current study, the mechanism has been examined by which two broadly expressed Dpp signaling target genes, optomotor-blind (omb) and brinker (brk), collaborate to initiate formation of the fifth longitudinal (L5) wing vein. omb is broadly expressed at the center of the wing disc in a pattern complementary to that of brk, which is expressed in the lateral regions of the disc and represses omb expression. A border between omb and brk expression domains is necessary and sufficient for inducing L5 development in the posterior regions. Mosaic analysis indicates that brk-expressing cells produce a short-range signal that can induce vein formation in adjacent omb-expressing cells. This induction of the L5 primordium is mediated by abrupt, which is expressed in a narrow stripe of cells along the brk/omb border and plays a key role in organizing gene expression in the L5 primordium. Similarly, in the anterior region of the wing, brk helps define the position of the L2 vein in combination with another Dpp target gene, spalt. The similar mechanisms responsible for the induction of L5 and L2 development reveal how boundaries set by dosage-sensitive responses to a long-range morphogen specify distinct vein fates at precise locations (Cook, 2004).

Extension of a previous analysis of ab in initiating L5 development has shown that ab functions early in L5 specification. Activation of all known vein genes, including rho, Dl, the caupolican and araucan genes of the Iroquois Complex (IroC), and argos, and repression of the intervein genes bs (also known as DSRF) and net, is lost in cells corresponding to the L5 primordium in ab1 mutant wing discs. A determination was also made whether it is critical that ab expression is confined to a narrow stripe for regulating expression of vein or intervein genes. ab was ubiquitously misexpressed in the wing disc using the MS1096-GAL4 driver; such global activation of ab suppresses expression of vein genes, such as rho and Dl. This ab misexpression also caused vein-specific downregulation of the intervein gene bs, in the wing disc, but did not repress expression of other genes, including hh, ptc and dpp. This phenotype may result from unregulated production of a lateral inhibitory signal normally produced by vein cells to suppress vein development in adjacent intervein cells (Cook, 2004).

Drosophila LSD1-CoREST demethylase complex regulates DPP/TGFβ signaling during wing development

The choice and timing of specific developmental pathways in organogenesis are determined by tissue-specific temporal and spatial cues that are acted upon to impart unique cellular and compartmental identities. A consequence of cellular signaling is the rapid transcriptional reprogramming of a wide variety of target genes. To overcome intrinsic epigenetic chromatin barriers to transcription modulation, histone modifying and remodeling complexes are employed. The deposition or erasure of specific covalent histone modifications, including acetylation, methylation, and ubiquitination are essential features of gene activation and repression. This study has found that the activity of a specific class of histone demethylation enzymes is required for the specification of vein cell fates during Drosophila wing development. Genetic tests revealed that the Drosophila LSD1-CoREST complex is required for proper cell specification through regulation of the DPP/TGFβ pathway. An important finding from this analysis is that LSD1-CoREST functions through control of rhomboid expression in an EGFR-independent pathway (Curtis, 2012).

The Su(var)3-3 gene (CG17149) encodes the Drosophila LSD1 homolog. Mutations in Su(var)3-3 result in aberrant histone methylation and heterochromatin formation, with increased global levels of H3K4me2 and impaired heterochromatic gene silencing. A physical association between LSD1 and CoREST has been described in Drosophila, revealing that the critical relationship between these proteins is conserved. LSD1 has an important role in organogenesis and germ line maintenance, such as during mouse anterior pituitary development and Drosophila ovary and wing development. LSD1 also regulates neural stem cell proliferation by modulating signaling via the orphan nuclear receptor TLX and LSD1 appears to have distinct functions in mammalian neuronal morphogenesis as well as stem cell self-renewal and differentiation. In humans, loss of LSD1 has been strongly correlated with several types of cancer and high-risk tumors, including prostate cancer, breast cancer and neuroblastomas). In contrast, overexpression of LSD1 has also been linked to some cancers. As a consequence of the emerging links between histone demethylase functions and disease, an understanding how LSD1 contributes to specific cell-cycle regulation and developmental processes is crucial (Curtis, 2012).

The Drosophila wing provides an outstanding in vivo model system to identify factors that regulate cell-fate determination as alterations in cell-fate can often be observed at the single cell level. Multiple conserved signaling pathways contribute to wing patterning and development and are regulated, in part, by the coordinated activities of chromatin remodeling complexes and epigenetic modifying enzymes. Previously work has identified histone lysine demethylase enzymes as coregulators of Brm complex remodeling activities in a genetic screen for factors that influenced a wing patterning phenotype associated with a conditional loss-of-function mutation in the snr1 gene that encodes a core regulatory subunit of the Brm complex. Genetic interaction tests indicated that lsd1 (Su[var]3-3) most likely interacted with the PBAP subtype of the Brm complex (Curtis, 2011). This report further addresses how LSD1 contributes to the cell-type and developmental time-point specific regulation of conserved signaling pathways by understanding its contribution to wing patterning and development (Curtis, 2012).

Recently, it was suggested that LSD1 regulates notch signaling during Drosophila wing development (Mulligan, 2011). This study presents evidence from genetic interaction analyses and tissue or cell-type specific targeted depletion experiments that suggest LSD1 and CoREST/CG42687 (synonymous with CG33525) may also regulate the DPP/TGFβ signaling pathway in a noncanonical manner, by regulating expression of rhomboid, a key player in canonical EGFR signal transduction. This is the first demonstration of LSD1-CoREST regulated DPP/TGFβ signaling and the results further define important roles of the LSD1-CoREST complex in tissue patterning (Curtis, 2012).

The appropriate elaboration of wing vein and intervein cell fates depends on the interplay of factors that promote and those that repress or block vein cell differentiation. In this study, we provide genetic evidence suggesting an important role for lsd1 and CoRest in repressing vein-promoting genes in intervein cells. Ectopic vein development can result from either the loss of a factor required for repressing vein cell differentiation or the gain of a factor that promotes vein cell fate in intervein cells. The experimental results suggest that lsd1 and CoRest utilize the first mechanism, since the aos hypomorphic mutation (aosw11), a factor known to repress vein fate, is enhanced by CoRestEY14216 and lsd1ΔN and targeted depletion by shRNAi of lsd1 and CoRest throughout the entire developing wing imaginal disc resulted in ectopic veins rather than loss of vein phenotypes. It was reasoned that if the LSD1-CoREST complex normally functions as a positive factor to promote vein development as proposed by the second mechanism, then mutations in lsd1 and CoRest or shRNAi depletion in the wing imaginal disc should produce a loss of vein phenotype. Based on the evidence presented in this manuscript, and on the recent finding that LSD1 is important for the regulation of NOTCH signaling in the wing (Di Stefano, 2011), it is proposed that the requirements of LSD1-CoREST are temporal and cell-type specific, and possibly dependent on the physical associations between LSD1 and several multiprotein complexes (Curtis, 2012).

An elaborate signaling network regulates wing patterning, where considerable cross-talk and functional redundancy connects five developmental pathways. For example, during pupal development, the main role of EGFR and DPP activation is to coordinately promote and maintain differentiation into vein cells while NOTCH activation establishes the provein-intervein boundary. However, DPP and NOTCH pathways are codependent, since expression of the NOTCH ligand, DELTA (DL) and its downstream target, ENHANCER OF SPLIT, (E(spl)mβ), require DPP signaling. LSD1 has been shown to interact directly with the histone deacetylase SIRT1 to repress NOTCH targets, suggesting important epigenetic functions for these co-repressors in metazoan development. However, recently it was shown that CoREST could function as a positive regulator of NOTCH in Drosophila follicle cells and wings (Domanitskaya, 2012). Therefore, there is growing precedent for the LSD1-CoREST complex to have both positive and negative roles in regulating gene expression depending on developmental context (Curtis, 2012).

LSD1 and CoREST depletion in the developing wing causes bifurcated or duplicated crossveins, a phenotype previously observed with Hairless (H) loss of function mutations. Because H both antagonizes NOTCH and promotes EGFR signaling, it is difficult to decipher the individual pathway regulated by LSD1-CoREST. Furthermore, the broadened vein delta phenotype observed at the wing margin in wing-specific LSD1-CoREST depleted and lsd1ΔN null flies (Di Stefano, 2011) is similar to Notch and DPP receptor (tkv) loss of function phenotypes (Curtis, 2012).

It is proposed that during the initial stages of wing vein development and differentiation, LSD1 negatively regulates NOTCH signaling. This is based on the observation that loss of lsd1 function suppresses the notched wing phenotype associated with mutations in suppressor of hairless (Su[HT4]) (Mulligan, 2011). However, later in development during vein refinement and maintenance, LSD1 appears to undergo a regulatory switch to positively regulate NOTCH signaling, since lsd1ΔN suppresses the short vein phenotype associated with the gain-of-function NAx-16 mutation. Additionally, the increased expression of downstream E(spl) targets in NAx-16 mutants is reversed by lsd1ΔN (Di Stefano, 2011). It was also recently shown that a transheterozygous mutant allele of CoRest (CoRestGF60) could enhance the wing phenotypes of flies carrying alleles of Dl and N (Domanitskaya, 2012), suggesting positive functions in regulating NOTCH signaling. Concurrently, LSD1 and CoREST repress vein cell differentiation by regulating components of the DPP signaling pathway at multiple points. For example, lsd1ΔN and CoRestEY14216 genetically interact with both dpp and genes encoding its receptors (e.g., dpp, tkv, sax), consistent with upstream functions. Strong genetic interactions were observed with downstream DPP signaling components (e.g., mad, med, ara, caup, shn), which suggests that the LSD1-CoREST complex has important regulatory functions in controlling the expression of DPP pathway targets. This conclusion is further supported by ectopic expression of the DPP-specific downstream signaling component, p-MAD, was observed in LSD1-CoREST-depleted animals. Activated DPP signaling is confined to proveins largely by the overexpression of TKV, a member of the TGFβ receptor family, in intervein boundary cells. TKV binds and sequesters the DPP morphogen. When TKV is downregulated, DPP spreads into regions of the wing destined to become intervein cells, resulting in ectopic veins. It is predicted that TKV is the most likely target of LSD1-CoREST complex regulation, since genetic interactions were observed between lsd1ΔN and CoRestEY14216 and almost all loss of function mutations in DPP signaling components, and tissue-specific LSD1-CoREST depletion lead to the development of ectopic veins, similar to phenotypes observed with loss of function alleles of tkv. Because activation of NOTCH and repression of DPP signaling are both required to repress vein promoting genes in differentiating intervein cells, LSD1 appears to have cell-type and context-specific activities to differentially regulate these pathways (Curtis, 2012).

Coimmunoprecipitation experiments suggested a complex forms between the HDAC1/2 class protein RPD3, LSD1, CoREST, and two TTK splice variants TTK88 or TTK69. Complexes containing CoREST/TTK69 or CoREST/TTK88 independently localize on polytene salivary glands, suggesting differential gene targeting. TTK and REST are likely functional homologs. Orthologs of tramtrack only exist in invertebrates, whereas REST orthologs are vertebrate-specific. TTK69 is a transcription factor that can recognize and bind to a specific DNA RE-1 consensus sequence (CCAGGACG), resulting in gene transcription. Unpublished observations suggest that TTK69, but not TTK88, function to negatively regulate vein cell development, since an incomplete vein phenotype is observed when TTK69 is overexpressed, whereas overexpression of TTK88 results in the development of ectopic veins. Therefore, it is predicted that LSD1-CoREST-TTK69 form a complex in developing wing tissue to negatively regulate DPP signaling in intervein cells. Furthermore, in mammals, the Brg1 complex chromatin remodeling capacity and recruitment specificity depends on formation of a LSD1-CoREST-REST-BRG1 comple. Because LSD1 can physically associate with the Brm chromatin remodeling complex in Drosophila, it is predicted that the Brm complex-LSD1-CoREST-TTK69 super-complex regulates genes essential for wing patterning, possibly through co-localization or recruitment to RE-1 consensus binding sites. Intriguingly, RE-1 consensus sites are present in both the rho and tkv gene loci, making these exciting targets for future investigation (Curtis, 2012).

Return: Rhomboid Transcriptional regulation part 1/3 | part 2/3

rhomboid: Biological Overview | Evolutionary Homologs | Protein Interactions | Developmental Biology | Effects of Mutation | References

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