rhomboid

Effects of Mutation or Deletion

Table of contents

Rhomboid and wing morphogenesis

There are a many wing vein mutants that interact strongly with rho. Based on these data a sequential model for wing veins can be devised. The formation of veins is a three step process. The first step, establishment of positional values requires engrailed, hedgehog, dpp, knot and others. Veins are formed at boundaries between positionally defined sectors. Of the genes involved in this step, rho interacts with en, hh, and kn. The second step is initiation of vein formation involving vein promotion genes including rho, drifter (also called ventral veinless), vein, and others. rhomboid interacts with vn and dfr. Vein suppression genes (net and plexus) are involved in initiation of vein formation. These genes interact with rhomboid. Additionally, genes involved in neurogenesis play a role in initiation of vein formation, and of these Serrate, Delta and Notch interact with rho. The third step in vein formation is a four stage process involving lateral inhibition to limit vein thickness, vein extention genes which assure continuity of veins, dorsal-ventral induction genes, which assure register of dorsal and ventral components of vein, and suppression of intervein genes which promote intervein fate. rhomboid interacts with thick veins and blistered (a suppression of intervein gene coding for the Drosophila serum response factor). In addition rho interacts with ventrolateral group genes and components of the ras signaling cascade including Star, Egf-r, ras1, gap1 and rolled. (Sturtevant, 1995).

Localized expression of the Drosophila rhomboid gene has been proposed to hyperactivate EGF-Receptor signaling in specific cells during development of the embryo and adult. Ectopic expression of a rho cDNA transgene causes dominant phenotypes, over half of which cannot be duplicated by ubiquitous expression of rho. Genetic interactions between various dominant ectopic enhancer alleles and mutations in the EGF-R/RAS signaling pathway indicate that many of these novel phenotypes result from ectopic activation of EGF-R signaling. Mis-expression of rho is sufficient for converting entire intervein sectors into veins (Noll, 1994).

The blistered (bs) locus has been examined phenotypically, genetically and developmentally using a set of new bs alleles. Mutant defects range from wings with ectopic veins and intervein blisters to completely ballooned wings where the distinction between vein and intervein is lost. rhomboid, a component of the EGF-R signalling pathway, is expressed in vein cells and is required for specification of vein cell fate. rhove mutations (lacking rhomboid in wings) suppress the excess vein formation and associated with bs. Conversely, rho expression in prepupal and pupal bs wings is expanded in the regions of increased vein formation (Fristrom, 1994).

How do veins on the dorsal and ventral surfaces of a fly's wing line up in exact opposition to one another? The adult Drosophila wing consists of two wing surfaces, apposed by their basal membranes, which first come into contact at metamorphosis, following wing disc eversion. Contact is crucial. Veins normally appear in these surfaces in a dorsal-ventral symmetric pattern, but when contact between the two surfaces is prevented, the dorsal-ventral pattern of venation takes on a 'corrugated,' asymmetric appearance (vein cells are more compacted and more pigmented). Dorsal-ventral contact apposition was prevented during wing imaginal disc morphogenesis by implanting fragments of discs into metamorphosing hosts. In these implants, longitudinal veins differentiate in both surfaces, but exhibit wider than normal corrugation. These results and those of genetic mosaics for mutants that remove veins or cause ectopic veins, reveal mutual dorso-ventral induction/inhibition at work to modulate the final vein differentiation pattern and/or corrugation. While clones of mutants causing a lack of veins (the single mutant rhomboid, the double mutant rhomboid vein, and the triple mutant extramachrochaetaeAch rhomboid vein) are autonomous in both wing surfaces, the vein phenotype is partially rescued by wild type cells from the opposite surface. When apposed to a lack of vein differentiation in the dorsal surface, vein differentiation fails in distal vein territories, preferentially in the ventral surface. Conversely, extra differentiation as in plexus, extramachrochaetae, HS-rhomboid 27B, Notch and Delta mutants, causes extra veins in clones, not only in the wing surface of the clone, but also in the opposite wing surface. Non-autonomous effects are observed in the same wing surface, a phenomenon called 'connectivity'. Genetic mosiacs of plexus72 and emc HS-rho27B cause neighboring non-mutant cells on the same surface to differentiate extra veins, connecting them. Cis (planar) and trans (vertical) effects may be operationally related, inducing contacting cells to differentiate vein histotypes. Vein cells induce vein differentiation in neighbouring cells, either on the same surface by planar cell-cell communication, or on the opposite surface through signals along the basal membrane of the apposing epithelium. Thus, although the vein pattern is surface-autonomously generated, inhibitory (negative) and inductive (positive) signals take place between both dorsal and ventral wing surfaces in order to refine the final vein pattern with the corresponding dorso-ventral wing surfaces (Milan, 1997).

In order to understand the role of blistered, coding for the Serum response factor, in wing development, blistered expression was examined in detail. The expression of a blistered reporter is first detected in imaginal wing discs by early third instar larva (70-80 hours AEL). At this stage, it is expressed homogeneously at low levels throughout the wing pouch, except in the presumptive wing margin. Mid-third instar imaginal wing discs (80-100 hours AEL) reveal increasing blistered levels, except in the wing margin; three perpendicular stripes of cells occur, corresponding to veins, where blistered expression begins to fade. At approximately the same developmental stage, veinlet (common alternative name rhomboid) is expressed in stripes corresponding to the gaps in blistered expression. The future veins L3, L4 and L5 will arise from these gaps. In late third instar imaginal wing discs (100-120 hours AEL), a further gap appears in the blistered expression, revealing the presence of the L2 vein. At this stage, a complex modulation of blistered expression is detected in the hinge region, possibly corresponding to the proximal vein trunks and interveins. There is also no expression in the notum. These gaps in the expression of blistered become more conspicuous in everted discs of pupae and, by 24-30 hours APF, all the interveins are apparent. blistered expression must be further refined after this stage, since the stripes lacking blistered are now about 6-8 cells wide, whereas in the adult wings they are only 3-5 cells wide. In adult wings, all vein cells lack blistered expression, which is present in all the intervein cells. The haltere is the only other imaginal disc to express blistered. blistered is expressed at high levels in the region corresponding to the pedicellum and scabellum, but is not present in the presumptive capitellum, the homologous region to the wing pouch. It is thus tempting to speculate that the absence of transalar connections and lack of apposition of dorsal and ventral surfaces in the haltere capitellum might be related to this non-appearance of blistered expression, such as occurs in the hollow wing veins (Roch, 1998).

The blistered function in wing vein development was examined by studying genetic mosaics of mutant cells, genetic interactions with other genes affecting vein development and blistered expression in several mutant backgrounds. Clones of blistered mutant cells proliferate normally but tend to grow along veins and always differentiate as vein tissue. These observations indicate that vein-determined wing cells show a particular behaviour that is responsible for their allocation to vein regions. Strong genetic interactions are observed between blistered, veinlet and genes of the Ras signaling cascade, in particular Egf receptor, rolled (rl) (MAPK), and a putative ligand of Egf receptor, vein, that codes for a neuregulin secreted protein. Hemizygosity for blistered totally suppresses the lack of vein L4 phenotype in Egfr, rolled and vein homozygous mutants, while greatly enhancing the amount of ectopic vein observed in a gain-of-function rolled allele. blistered hemizygosity also suppresses the lack of veins in veinlet hypomorph conditions. Conversely, it dramatically enhances the amount of ectopic vein tissue obtained after ubiquitous expression of veinlet + product (Roch, 1998).

The observed interaction between Egf receptor and blistered in hypomorphic conditions led to the study of double mutants for strong alleles of both genes in mosaic clones. Double mutant clones were generated at 48-72 hours AEL for the top 4A and bs P1292 alleles. top 4A clones appear with a reduced frequency, are smaller, narrower and more elongated than controls, and are composed of small cells that are unable to differentiate vein histotype, leaving a gap of intervein tissue wherever they touch a vein, except in the anterior wing margin vein (L1). Double mutant top 4A;bs P1292 clones tend to occupy vein territories like bs P1292 clones, a preference never observed in top 4A clones, but one which appears with a frequency and size similar to top 4A controls. Double mutant cells differentiate autonomously, in all cases, into pigmented, corrugated and compacted tissue with smaller cells than those characteristic of torpedo. The observation of these typical vein features leads to the conclusion that this tissue has a vein histotype indicating that the blistered extra vein phenotype is epistatic to torpedo (Egfr) lack of veins. It is concluded that during disc proliferation, blistered expression is under the control of the Ras signal transduction pathway, but its expression is independent of veinlet. During the pupal period, blistered and veinlet expression become interdependent and mutually exclusive. These results link the activity of the Ras pathway to the process of early determination of intervein cells, by the transcriptional upregulation of the blistered nuclear factor (Roch, 1998).

The function of extra macrochaetae is required during wing morphogenesis. Mitotic recombination clones of both null and gain-of-function alleles of emc, indicate that during wing morphogenesis, emc participates in cell proliferation within the intervein regions (vein patterning), as well as in vein differentiation (de Celis, 1995). The study of relationships between emc and different genes involved in wing development reveal strong genetic interactions with genes of the Ras signaling pathway (torpedo, vein, veinlet and Gap), and with several other genes (blistered, plexus and net) in both adult wing phenotypes and cell behaviour in genetic mosaics. These interactions are also analyzed as variations of emc expression patterns in mutant backgrounds for these genes. In addition, cell proliferation behaviour of emc mutant cells varies depending on the mutant background. The results show that genes of the Ras signaling pathway are co-operatively involved in the activity of emc during cell proliferation, and later antagonistically during cell differentiation, repressing EMC expression (Baonza, 1999).

Evidence that emc acts co-operatively with genes of the Ras pathway consists of studies of wing size in flies with multiple mutations. top¹ homozygous mutant wings, mutant for the gene coding for the Epidermal growth factor receptor, are 14%-20% smaller than wild type, whereas top;emc double mutant wings are 36%-42% smaller. Surprising, the interaction between loss of function (LOF) alleles of emc and hypomorphic alleles of top and vein (vn) produces the same effect on reduction of wing size as the interaction between gain of function (GOF) hypermorphic emc and mutations in these genes. Thus, the vn wings are 15% smaller than the control wings, and in combination with the LOF and GOF alleles of emc the wings appear 27%-35% and 21%-29% smaller than wild type wings, respectively, suggesting that GOF alleles may have a LOF component (Baonza, 1999).

Evidence that emc acts antagonistically to Ras pathway genes during vein differentiation consists of observations of vein differentiation in genetic mosaics. LOF alleles of member genes of the Ras pathway exhibit the absence of veins, and conversely, mutations that cause an increase in the activity of that pathway result in the appearance of ectopic veins. Mutant vein phenotypes of LOF alleles of genes of the Ras pathway are suppressed in interactions with the LOF alleles of emc and enhanced with the GOF allele of emc. Reciprocally, the extra veins mutant phenotype of the GOF alleles for genes of the Ras pathway, is increased in interaction with LOF alleles of emc and reduced with the GOF allele of emc, indicating an antagonistic relationship between emc and genes of the Ras pathway. LOF alleles of emc rescue the lack of vein differention in emc;ve;vn triple mutant clones (ve is veinlet, coding for the protein better known as Rhomboid). Contrarily, double mutant clones of emc and alleles of members of the Ras pathway, which correspond to a hyperactivation of this pathway (Gap1 or heat shock rhomboid), differentiate ectopic veins everywhere in the wing vein (Baonza, 1999).

Genetic interactions have also shown synergistic mutant effects on venation between emc, plexus (px whose molecular nature is unknown) and net, which codes for a bHLH transcription factor. The net gene is required for intervein fate in wings. Furthermore, emc expression, which is absent in normal veins, also disappears in pupal extra veins caused by px and net. Given the molecular nature of net, the co-operative behavior wth emc could reflect direct molecular interactions. Similarly, genetic interactions and changes in expression pattern of emc are found with blistered (bs) mutants. blistered, coding for the Serum response factor of Drosophila, is expressed in the future intervein issue of the wing imaginal disc, in a complementary pattern to Ras pathway genes. In wing differentiation, bs plays a dual role in wing development. Two fully active copies of bs are required to ensure that the formation of wing veins is limited to vein territories. In addition Bs protein is essential for proper terminal differentiation of intervein cells. bs causes strong phenotypic interactions with mutants of the Ras pathway. Thus, it is proposed that emc, bs, px, net and the Ras signaling pathway set of genes are intimately related in vein/intervein patterning and differentiation. The Ras signaling pathway is thought to be involved in maintaining low levels of emc expression during vein pattern differentiation in cells that will differentiate as veins. This is consistent with observations of the expression pattern of emc. Emc protein and mRNA are found at highest levels in intervein regions (Baonza, 1999).

Genes of the ventrolateral group in Drosophila are dedicated to developmental regulation of Egfr signaling in multiple processes including wing vein development. Among these genes, Egfr encodes the Drosophila Egf-Receptor; spitz (spi) and vein (vn) encode EGF-related ligands, and rhomboid and Star (S) encode membrane proteins. This study shows that rho-mediated hyperactivation of the Egfr/MAPK pathway is required for vein formation throughout late larval and early pupal development. Consistent with this observation, rho activity is necessary and sufficient to activate MAPK in vein primordia during late larval and early pupal stages. Epistasis studies using a dominant negative version of Egfr and a ligand-independent activated form of Egfr suggest that rho acts upstream of the receptor. rho and S function in a common aspect of vein development since loss-of-function clones of rho or S result in nearly identical non-autonomous loss-of-vein phenotypes. Furthermore, mis-expression of rho and S in wild-type and mutant backgrounds reveals that these genes function in a synergistic and co-dependent manner. In contrast, spi does not play an essential role in the wing. These data indicate that rho and S act in concert, but independent of spi, to promote vein development through the Egfr/MAPK signaling pathway (Guichard, 1999).

rho and S were initially identified based on similar embryonic loss-of-function phenotypes, suggesting that they are involved in a common molecular process. Moreover, S is the most potent known dominant suppressor of rho-induced extra-vein phenotypes. Further support for a close partnership between these two genes has been provided in this study. In the wing, loss-of-vein phenotypes caused by rho- or S- clones are very similar, but different from phenotypes associated with clones of mutants in the Egfr signaling cassette. For example rho- and S- clones exhibit local cell non-autonomy, in contrast to Egfr - clones. Furthermore, cells in rho - or S - clones have normal viability and size, whereas mutant clones lacking Egfr or downstream components have reduced cell size and viability. Thus, loss-of-function analysis reveals that rho and S define a subgroup of genetic functions required for strong EGFR signaling, distinct from activities in the EGFR/MAPK pathway proper (Guichard, 1999).

The relationship between rho and S was addressed in series of epistasis experiments. Overexpression of S, which alone is unable to cause any phenotype in the wing, generates a strong ectopic vein phenotype only when ectopic rho is provided, showing that S needs rho to function in intervein regions. Clonal analysis indicates that S also depends on endogenous rho expression in veins, since overexpression of S in the absence of rho cannot rescue vein formation. Reciprocally, strong ectopic rho expression cannot generate any phenotype in wing clones lacking S. Collectively, these data indicate that rho and S function co-dependently, and collaborate to activate Egfr signaling by a common molecular mechanism (Guichard, 1999).

Although rho and S mutant phenotypes are similar during many stages of development, the co-dependence of rho and S does not seem to apply to the eye. In the eye imaginal disc, S is required for cell viability and generates dominant morphological defects in the heterozygous condition, while clones lacking rho have no obvious phenotype. This difference between rho and S function in the embryo and wing, versus in the eye, could be explained by the possible existence of other Rho-like proteins interacting with S during eye development, or might reflect the ability of S to function in the absence of Rho in certain cellular contexts (Guichard, 1999 and references).

Two classes of models have been proposed to explain the activity of Rho at the molecular level. In the first type of model, Rho activates a separate signaling pathway that ultimately converges on the RAS/MAPK pathway. This class of models accounts for the fact that in most situations, rho is required in the cells in which it is expressed. An exception to this rule is in embryonic chordotonal organs, where rho is expressed in the sensory organ precursor cell, but activates MAPK only in surrounding epidermal cells. In a second class of models, Rho produces an extracellular signal that activates Egfr. The current data are consistent with aspects of both models, but do not support the specific proposal that Rho promotes the processing of an m-Spi precursor into a diffusible active form. According to this latter model, spi- loss-of-function clones would be expected to induce phenotypes equivalent to or stronger than those observed in rho- or S- clones, particularly in a vn¹ mutant background. Since spi- clones have no detectable effect, the data argue against the 'Spi processing' model in the context of wing vein development. Even if Spi produced in wild-type cells were able to diffuse and rescue the vein-loss phenotype of spi- clones, the fact that small rho - or S - clones induces vein-loss phenotypes argues strongly against the possibility that Rho acts through the processing or the activation of m-Spi. In contrast, the ability of m-Spi to enhance and sharpen the vein phenotype caused by ectopic Rho and Star proteins suggests that Rho and Star can collaborate with m-Spi to generate a stronger and more localized signal. The inability of m-Spi to induce any phenotype in the absence of ectopic Rho and Star proteins suggests that m-Spi may require a prior action of Rho and Star to activate the Egfr in this artificial situation, whereas s-Spi or Vein do not. It is also very unlikely that Rho promotes the processing of Vein, since the Vein ligand is not transmembrane bound and does not require a cleavage to function. Nevertheless, it is possible that Rho and S support the processing or facilitate the presentation of an unknown Egf ligand in the wing, or promote transcytosis of Egf ligands or ligand-receptor complexes. The drawback to these last two hypotheses is that they involve an as yet unidentified molecule(s). Alternatively, S may constitute the missing link between Rho and Egfr. S could be a precursor for a diffusible factor, which ultimately activates Egfr, either as a ligand, or as a co-ligand. Such a co-ligand would reinforce or coordinate the effect of EGF ligands, possibly by promoting the formation of receptor oligomers. According to this scenario, Rho might act by promoting the processing of a membrane tethered S precursor into an active diffusible form. Finally, Rho and S could be acting directly on the Egfr (i.e. on the extracellular domain of the receptor itself) of the cells expressing Rho, and sometimes also of the adjacent cells (e.g. Rho could promote receptor dimerization or aggregation and thereby enhance the Egfr signal). Although this last hypothesis would not account for rho action over more than one cell diameter, it could explain why rho generally has a greater effect and ability to promote MAPK activation in cells expressing rho. Additional biochemistry experiments will be required to understand the basis for the non-autonomous action of Rho, and its predominant action in cells in which it is expressed (Guichard, 1999 and references).

A screen was carried out in order to identify genes interacting with Armadillo, the Drosophila homolog of ß-catenin. Two viable fly stocks have been generated by altering the level of Armadillo available for signaling. Flies from one stock overexpress Armadillo (Arm^over) and, as a result, have increased vein material and bristles in the wings. Flies from the other stock have reduced cytoplasmic Armadillo following overexpression of the intracellular domain of DE-cadherin (Arm^under). These flies display a wing-notching phenotype typical of wingless mutations. Both misexpression phenotypes can be dominantly modified by removing one copy of genes known to encode members of the wingless pathway. This paper identifies and describes further mutations that dominantly modify the Armadillo misexpression phenotypes. These mutations are in genes encoding three different functions: establishment and maintenance of adherens junctions, cell cycle control, and Egfr signaling (Greaves, 1999).

Mutations in 17 genes (26 deficiencies) were characterized that interact with Arm^over and/or Arm^under. Interaction strength varies from deficiency to point mutation, suggesting that several genes in the original deficiencies could have contributed to, or modified, the interaction. Only for 7 of the 17 genes have interactions been identical between the point mutation and the corresponding starting deficiency. The 17 genes were sorted into four groups. Group 3 consisted of EGF pathway genes: Interactions have been observed with some, but not all, members of the EGF pathway. Those identified were Egfr, veinlet/rhomboid (ve/rho), and argos (aos). All enhance Arm^over, increasing the number of ectopic bristles in the wing blade, with ve^M4 being the strongest interactor. None show an interaction with the Arm^under stocks.

The interactions with genes encoding components of the Egf pathway were initially dismissed because argos and Egfr, which have opposite effects on the Egf pathway, interact similarly with Arm^under and Arm^over. However, recent work has demonstrated an antagonism between the Wg and Egf pathways in the embryonic epidermis. This antagonism is probably not universal since another embryonic function of Wg, the maintenance of Engrailed expression, is not affected by Egf signaling. However, the interactions that were uncovered in the wing suggest that the Egf-Wg antagonism may not be limited to cuticle patterning. It is noteworthy that, in the wing, only an interaction with Arm^over (which involves ectopic bristles) was seen. It may thus be that Wg and Egf only compete at places where specialized cuticular structure are formed, although, while denticles are negatively regulated by Wg, bristles are made in response to Wg signaling. No explanation is available as to why argos, a negative regulator of Egf signaling, should interact in the same manner as Egfr (Greaves, 1999).

Wing vein development in Drosophila is controlled by different morphogenetic pathways, including Notch. Hairless (H) antagonizes Notch target gene activation by binding to the Notch signal transducer Suppressor of Hairless [Su(H)]. Accordingly, overexpression of H phenocopies reduction of Notch activity. In the construct H-C2, the presumptive Su(H)-binding domain of Hairy has been removed. As a consequence, H-C2 protein has completely lost the ability to bind to Su(H) protein and to interfere with Su(H)-dependent developmental processes like bristle development, wing margin specification or vein width refinement in vivo. Apart from the internal deletion, the H-C2 protein compares to the wild type with respect to antibody recognition, apparent molecular weight, subcellular distribution, stability as well as biochemical interactions with other H partner proteins. With regard to endogenous activity, the H-C2 lines are rather weak compared to the full length H constructs, however, after heat shock induction, expressivity is similar. Surprisingly, overexpression of H-C2 induces lethality like the wild type construct and in addition, leads to the induction of ectopic vein material in certain intervein regions of the wing (Johnnes, 2002).

Keeping in mind that H itself is not a transcriptional regulator but rather functions through protein-protein interactions with different protein targets, these phenotypes cannot be simply explained by altered activation of Notch target genes. Rather, they might uncover a currently unknown Su(H) independent activity of H involving different protein(s) maybe outside of the Notch signaling cascade. In order to understand this phenomenon, the involvement of H-C2 in the process of vein establishment was analyzed in comparison to wild type H. The data presented in this work are in agreement with a model whereby H, apart from antagonizing Notch signaling, positively regulates EGF signaling during the process of wing vein formation (Johnnes, 2002).

In a screen for genetic modifiers of the H-C2 phenotype, several genes involved in Notch and epidermal growth factor (EGF) signaling were identified. Most notably veinlet, an activator of EGF signaling, acts downstream of H-C2. H-C2 positively regulates veinlet maybe through inhibition of intervein determinants in agreement with a model, whereby Notch and EGF signaling pathways cross-regulate vein pre-patterning (Johnnes, 2002).

Overexpression of hs-H-C2 induces ectopic veins only between day 5 and 6 after egg deposition. Phenotypes vary significantly and were arranged into a phenotypic series of five classes. Using precisely synchronized cultures, the pheno-critical period was restricted to pre-pupal and early pupal developmental stage, starting approximately at the larval-prepupal transition. Induction of H-C2 during mid- to late-third instar larval stages did not result in ectopic vein formation even with prolonged and, in their consequence, semi-lethal heat shocks. Ectopic venation was not randomly distributed and certain regions of the wing blade were more sensitive than others. In order to distinguish between temporal and/or sensitivity differences, the wing was partitioned into distinct intervein sectors A-F and the appearance of extra veinlets over time for each sector was scored independently. As became apparent, the six different sectors respond with a similar temporal profile, but with different sensitivities. The less sensitive sectors A and C revealed two pheno-critical periods, which might, however, be a consequence of the time convolution of the data. In summary, the main impact of H-C2 on the wing venation process occurs in the pre-pupal and early pupal stages of development (Johnnes, 2002).

The H-C2 protein is unable to bind to Su(H) and has lost basically all of H wild type activity: H-C2 is unable to rescue the haplo-insufficient H loss-of-function phenotype and does not cause the typical H gain-of-function bristle phenotypes. Thus, H-C2 venation phenotypes might either uncover a Su(H) independent function of H or an unrelated, novel activity. Although overexpression of H wild type constructs causes only little ectopic vein material, H and H-C2 proteins are able to synergize upon overexpression. It is concluded that the vein inductive property of H-C2 is a native function of H, and that H is able to partially substitute for H-C2 in this process (Johnnes, 2002).

If indeed the vein inductive property of H-C2 uncovers a Su(H) independent activity of H, the question arises as to what other targets H might act upon. In order to identify such putative targets, a candidate screen was set up for dominant modifiers, concentrating on the three main signaling pathways which normally contribute to wing vein development: Notch, Egf and Dpp signaling cascades. In the double heterozygotes, H-C2 was induced during the pheno-critical period with double heat shocks to make up for the weak phenotypes caused by a single hs-H-C2 copy (Johnnes, 2002).

Components of the EGF signaling pathway, screened for dominant interactions with H-C2, included ligand and its processing, the receptor itself, components of the signal transduction cascade as well as related genes. As expected, ve and to a lesser degree Star (S) mutations dominantly reduce the amount of extra vein material. Both genes are essential for vein formation, and thus, reduction of their activity was expected to antagonize the H-C2 vein-promoting effect. By lowering the ve gene dose, the suppression is almost complete except for some ectopic vein material in the region of the anterior cross-vein, a region which is not affected by the homozygous ve¹. Only weak, interactions were found with Dpp pathway mutations (Johnnes, 2002).

In agreement with the essential role of ve in the establishment of vein fate, data indicated that the homozygous ve¹ mutant completely suppresses ectopic vein induction through hs-H-C2, except for some small veinlets. No ectopic veins were visible in the distal wing blade, where the ve¹ phenotype is apparent, even after strong overexpression. This demonstrates that H-C2 strictly depends on ve for the induction of veins and suggests that overexpression of H-C2 might somehow result in the ectopic activation of ve (Johnnes, 2002).

In accordance with the proposed role of net as negative regulator of ve, net¹ mutations cause extensive extra veins. This phenotype comprises nearly all aspects of hs-H-C2 overexpression in a wild type background. Unexpectedly, overexpression of H-C2 enhances the net phenotype considerably: not only do the heterozygous net¹ mutants resemble the homozygotes, but the homozygotes developed massive patches of vein tissue and extensive blistering of the wing. Moreover, net¹; hs-H-C2 homozygotes were semi-lethal at 25°C and the stock was only viable at 18°C. Because net¹ is a complete null allele, this result excludes the simple model that H-C2 promotes vein development by inhibition of net activity. Instead, H-C2 acts independent of net either as a vein-promoting factor, e.g., by activation of ve, or by repression of other negative regulators of ve that act in addition to net. The latter seems more plausible with regard to normal H function. In the heterozygous net¹ background, which is phenotypically wild type but sensitized for ectopic vein formation, the vein-promoting activity of H is revealed: overexpression of full length H in net¹ heterozygotes results in ectopic venation that is a perfect phenocopy of the H-C2 effects. Apparently, wild type H has a vein-promoting activity, which also can be explained by antagonizing a negative regulator of ve (Johnnes, 2002).

Since induction of ectopic vein material by H-C2 is extremely sensitive to developmental time, the epistatic relationship with ve¹ was reassessed by continuously overexpressing H-C2 or H with the aid of the Gal4/UAS system in a wild type and a ve¹ background. Prolonged overexpression of H-C2 with en-Gal4 or Bx^MS1096-Gal4 driver lines results in conversion of most of the distal intervein areas to vein tissue. Only the region between L3 and L4 proved resistant. Furthermore, induction of microchaetae in this area was observed. Apart from this conversion, extensive tissue loss was noted while the wing margin itself remained intact. Overexpression of H-C2 in the ve¹ background shows both tissue loss and ectopic microchaetae on the wing blade. Again, no induction of ectopic vein material was observed in the more distal regions in support of the notion that H-C2 acts upstream of ve. It is proposed that H-C2 promotes vein induction by up-regulating ve activity. Moreover, processes independent of ve are influenced by H-C2, which finally lead to the induction of ectopic bristles on the wing blade and to tissue loss. Overexpression of H in the same experimental set-up was unable to convert intervein tissue into vein material, apart from few ectopic veinlets. At the same time, H causes wing tissue and margin loss accompanied by broadened wing veins. These are the known hallmarks of an impaired Notch signaling (Johnnes, 2002).

Since vein determination depends on the balance between ve and bs activity, the influence of H or H-C2 on wild type bs or ve gene expression was examined. Full length H and H-C2 expression was ectopically induced from UAS-constructs in the posterior compartment via the en-Gal4 driver. ve expression was monitored with either enhancer trap line, ve^rho-lac1 or ve^X81. Expression of ve and bs is mutually exclusive in vein and intervein tissue, respectively. Overexpression of H does not alter this complementary expression pattern, however, overexpression does cause strong ve expression within and confined to pro-vein areas. This is in contrast to the effects of H-C2 overexpression where ectopic expression of ve^rho-lac1 is very prominent in pupal wings already in early pupal stages and remains strongly activated at least until 36 h after pupal formation (APF). Earlier wing development was analyzed using the ve^X81-lacZ reporter. In wing discs from late third instar larvae, no patterning defects apart from tissue loss are observed. However, deviations from wild type become apparent already in the pre-pupal wing discs as early as 4 h APF. At 6 h APF, a strong and reliable ectopic staining near the wing margin, where pro-vein 5 develops, is observed that later spreads into the adjacent intervein field. Overall, ectopicve expression reliably predicts the pattern of ectopic venation caused by overexpression of H-C2. This is in agreement with the hypothesis that ve regulation is a target of H-C2 activity (Johnnes, 2002).

The simplest model to explain the differential effects of H and H-C2 on the regulation of ve expression would be the assumption of a vein inductive role of Notch in pre-pupal and early pupal development in agreement with the biphasic developmental role of Notch, e.g., during eye development, where, in the course of lateral inhibition, Notch first promotes proneural fate before restricting this fate to single photoreceptor precursor cells. By binding to Su(H), H would limit such a vein-promoting activity of Notch at an early inductive phase. Because H-C2 is unable to bind to Su(H), an assumed inductive Notch signal would be able to pass and thus, set the stage for ectopic veins. To test this assumption, an activated Notch receptor (N^intra) was expressed under heat shock control during the H-C2 pheno-critical period. Since hs-N^intra overexpression at 37° proved extremely lethal to larvae and pupae alike, the induction was performed at lower temperatures of 34-35°. Under these conditions, hs-N^intra is able to induce ectopic veinlets, mostly of cross-vein character, in all the regions where H-C2 is also able to induce ectopic vein material. These results demonstrate that Notch signaling is able to exert a positive influence on wing vein specification during early pupal stages, closely followed by the well characterized vein suppressing activity of Notch signaling (Johnnes, 2002).

The onset of pupariation is a major developmental switch, where expression of many genes as well as their developmental effects changes dramatically. This is, for example, observed in the regulation of ve and bs from third instar larval stage to early pupal stage: whereas the activation of both genes early on depends on pre-pattern genes like net, their regulation becomes inter-dependent and mutually exclusive about 4 h after puparium formation. The abrupt onset of H-C2 vein-promoting activity is interpreted accordingly. Maybe, H-C2 responds to or influences the activity of other factors which only become available at that time and play a role in the promotion or repression of vein development. This is reflected by the onset of the positive influence of H-C2 on ve expression at around 4 h after pupariation. Thus, the H-C2 pheno-critical period might reflect a developmental switch for a requirement of H activity for vein fate decisions (Johnnes, 2002).

Involvement of the Notch signaling pathway in the refinement of proper vein width is well established. Current models suggest that during this lateral inhibition process, Dl acts as an inhibitor of vein formation by directing cells, neighboring presumptive vein cells, into the intervein fate. This model is in line with the observation that Dl mutants act as enhancers of H-C2 vein promotion. In Dl mutants, the threshold for vein fate is lowered as determined vein cells are less likely to be driven back into intervein fate. The interrelationship of H-C2 and Notch signaling during vein formation is, however, not restricted to the process of vein width refinement. Overexpression of N^intra promotes early vein formation and may thus be setting the stage for pro-vein development within intervein areas. Despite the fact that Notch activity is not necessary for pro-vein specification itself, it is required for the vein-promoting activity of H-C2 because Notch mutations act as strong dominant suppressors of H-C2 effects. In agreement, N^Ax-E2, a hyper-activated allele of Notch, acts as a weak enhancer of H-C2 and it is even possible that this effect is initially stronger but then obliterated through enhanced lateral inhibition (Johnnes, 2002).

Reduction of the ve gene dose results in a very pronounced, dominant repression of the H-C2 phenotype despite the fact that the allele ve¹ has no dominant visible phenotype. This suggests that ve plays a crucial role for H-C2 to exert its inductive effects. Interestingly, dosage reduction of either the Drosophila EGF receptor or the MAPK rolled (rl) has no dominant influence on the H-C2 ectopic venation phenotype. The former result was unexpected and suggests that the Drosophila EGF-receptor itself is not rate limiting in this process. This notion is in line with the observation that also ve¹ is fully recessive in combination with loss-of-function alleles for the Drosophila EGF-receptor. The allele rl¹ is a mild hypomorph and the reduction of MAPK activity might not be strong enough to influence the H-C2 phenotype. Together with the results of full epistasis of ve over H-C2, these data suggest that neither the Drosophila EGF-receptor nor the EGF signal transduction cascade are influenced by overexpression of H-C2 (Johnnes, 2002).

Overexpression of H results in the extension of ve expression all over the pro-vein area, whereas overexpression of H-C2 induces, in addition, ve outside the pro-veins, also within the intervein fields. Thus, both act positively on ve regulation but H-C2 is clearly different from H with respect to the apparent conversion of presumptive intervein- to pro-vein cell fates. Pro-vein activity is a normal aspect of H wild type function that is uncovered in a sensitized background: halving the gene dose of net or bs might result in a subtle increase of ve activity which can then be pushed by H above the threshold for pro-vein fate. Although the results clearly demonstrate that H and H-C2 act positively on the regulation of ve, it cannot be concluded that this regulation is direct. Rather H might act negatively on the output of vein repressing factors. Since H has the capability to interact with a number of different proteins, overexpression of either H or H-C2 could influence stoichiometry of complexes or availability of factors involved in ve regulation. Two such factors that have overlapping activity with regard to the negative regulation of ve are encoded by net and bs, however, they show remarkably different temporal activity profiles in that net acts during larval stage and bs at the transition of larval to pre-pupal stage. Thus, bs appears the more likely target of H activity; this is supported by the fact that H-C2 can promote vein induction even in the absence of net and that bs repression is observed at the anterior cross-vein without simultaneous ve induction. Whether this inhibition is direct or via as yet unknown factors and pathways requires further study (Johnnes, 2002).

Thus, both H and H-C2 have the ability to up-regulate ve. However, in contrast to H, H-C2 overexpression is capable of overriding intervein fate and thus inducing ve expression within intervein territories. Several lines of evidence suggest that this is a normal facet of H function. (1) Synergistic effects of combined overexpression of H and H-C2 were found. (2) In a sensitized genetic background of reduced copies of intervein specifying genes like bs and net, H itself possesses vein-promoting activity just like H-C2. These results can be explained if one assumes a dual, independent activity for H: in one case, H might up-regulate ve, for example, by interfering with intervein specifying factors, an activity retained by H-C2. In another case, H, by virtue of binding to Su(H), antagonizes Notch-dependent processes such as an early vein fate-promoting activity and subsequently vein width refinement. This activity is presumably lost in H-C2 due to the lack of the Su(H)-binding domain. Unfortunately, attempts to directly test this hypothesis failed because Su(H) mutant clones could not be generated in the background of H-C2 (Johnnes, 2002).

A dual role of H in suppressing inductive Notch signaling and enhancing ve activity would explain why the wild type protein is unable to induce ectopic venation except in a sensitized background, where ve activity has already reached a critical threshold by the reduction of its negative regulators. In contrast, H-C2 can no longer antagonize Notch activity, but might still promote vein formation by interfering with ve suppression. Altogether, the genetic data may be taken as an example for a link between Notch and EGF signaling, which in the context of vein formation appears to involve the activity of H influencing both Notch signaling and, via ve, EGF signaling as well (Johnnes, 2002).

Drosophila wing development is a useful model to study organogenesis, which requires the input of selector genes that specify the identity of various morphogenetic fields and cell signaling molecules. In order to understand how the integration of multiple signaling pathways and selector proteins can be achieved during wing development, the regulatory network that controls the expression of Serrate (Ser), a ligand for the Notch (N) signaling pathway, which is essential for the development of the Drosophila wing, as well as vertebrate limbs, was examined. A 794 bp cis-regulatory element located in the 3' region of the Ser gene can recapitulate the dynamic patterns of endogenous Ser expression during wing development. Using this enhancer element, Apterous (Ap, a selector protein), and the Notch and Wingless (Wg) signaling pathways, are shown to sequentially control wing development through direct regulation of Ser expression in early, mid and late third instar stages, respectively. In addition, later Ser expression in the presumptive vein cells is controlled by the Egfr pathway. Thus, a cis-regulatory element is sequentially regulated by multiple signaling pathways and a selector protein during Drosophila wing development. Such a mechanism is possibly conserved in the appendage outgrowth of other arthropods and vertebrates (Yan, 2004).

The results reported here demonstrate that a 794 bp cis-acting regulatory module in the Ser locus can be temporally regulated by three distinct mechanisms that are employed for the proper establishment of the DV organizer during wing development. (1) The selector protein Ap directly activates Ser expression in the dorsal compartment during the early third instar, which sets up N activation for the next stage. (2) By the middle of the third instar, the N pathway maintains Ser expression by a positive-feedback loop along the DV boundary. This feedback loop maintains Ser and Dl expression, leading to the activation of N signaling at the DV boundary, which is essential for establishing the DV organizer. (3) At the end of the third instar, as a result of Wg signaling, Ser is expressed in two stripes flanking the DV boundary, which limits N activation to the DV border. In addition, Ser expression in provein cells is dependent on input from the Egfr pathway. These results indicate how tissue-specific selector and signaling molecules can work sequentially to achieve a complex developmental process, such as organogenesis, which involves a complex temporal and spatial regulation of genes. However, the conclusion that the Ser minimal wing enhancer is sequentially regulated by Ap, Notch, Wg and Egfr does not exclude the possibility that these molecules/signaling pathways may cooperate and synergistically stimulate gene expression at certain stages. In this case, mutations that specifically impair response to the intended factor would affect Ser-lacZ expression in other phases of disc development (Yan, 2004).

Ser is expressed in provein cells and its expression is regulated by Egfr signaling at the pupal stage. N signaling also plays an important role in determining vein cell fate. The data on Ser expression in provein cells is consistent with a report on Ser function during vein development. Thus, in addition to its essential role in development of the Drosophila leg and vertebrate limbs, Egfr/Fgf signaling also plays a role in Drosophila wing development, suggesting a conserved role of Egfr signaling in 'appendage' development. Ser expression was examined in both gain-of-function (gof) and loss-of-function (lof) Egfr signaling-mutant backgrounds. First, in a rho gof mutant (UAS-rho^*), Ser appeared to be ectopically expressed between L3 and L4, exactly where ectopic rho activity was localized. Ser expression in the proveins was eliminated in a rho and vein (vn, encoding a Egfr ligand) double-mutant (Egfr lof) background, in which vein formation is completely abolished. These results suggest that the Egfr pathway may regulate Ser expression during vein development at the pupal stage. Interestingly, the Ser minimal wing enhancer is expressed in provein cells at both larval and pupal stages. Further investigation of this element may shed light on how Egfr signaling regulates vein differentiation (Yan, 2004).

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