Transcriptional Regulation

The ventralized phenotype of dodo is reminiscent of that of the hypomorphic Egfr- mutant or of mutants ectopically overexpressing CF2 (via a heat-shock promoter). As with the Egfr- and hs-CF2 mutants, dodo mutation also results in maternal-effect embryonic lethality; that is, the progenies of dodo females show early patterning defects even when mated with wild-type males. The embryonic defects are typically expressed in the mutant cuticles as expanded and fused ventral denticles. CF2 degradation in the dorsal follicle cells no longer occurs in the dodo deletion mutant. The repression target of CF2, the rhomboid gene, consequently also exhibits a ventralized expression pattern. Consistent with the variable severity of the eggshell defects, rhomboid expression either is completely abolished or shows a fused two-stripe pattern, the latter reflecting perfectly the fused-appendage phenotype. The variability of rhomboid expression also indicates that the penetrance of the dodo mutant is not 100% (Hsu, 2001).

Rhomboid and Epidermal growth factor receptor signaling are involved in the process of segmentation. The spread of Wingless within the embryonic epidermis of Drosophila was examined in order to understand the developmental biology of segmentation. Using two assays for Wingless activity (specification of naked cuticle and repression of rhomboid transcription), it was found that Wingless acts at a different range in the anterior and posterior directions. This asymmetry follows in part from differential distribution of the Wingless protein. Transport or stability is reduced within engrailed-expressing cells, and farther posteriorward Wingless movement is blocked at the presumptive segment boundary and perhaps beyond. The role of hedgehog in the formation of this barrier is demonstrated (Sanson, 1999).

It is proposed that asymmetric Wingless distribution ensures the establishment of well-differentiated cell fates on either side of the engrailed domain. Anteriorly, at the wingless source, rhomboid expression is repressed. In contrast, reduced Wingless movement and/or stability within the engrailed domain allows nascent posterior rhomboid expression. Around this time (stage 11), a barrier to Wingless that requires hedgehog signaling forms at the posterior of the engrailed domain and ensures that Wingless does not foray across and repress rhomboid. rhomboid then activates the Egfr pathway within its expression domain and in adjacent cells. It may be that rhomboid itself contributes to barrier formation and thus builds a line of defense against invasion by its repressor. In addition, activation of the EGF pathway by rhomboid would antagonize any Wingless leaking through. Denticle formation requires transcription of shavenbaby, which is under positive regulation by the Egfr pathway and negative regulation by the wingless pathway. Activated EGFR and the absence of Wingless posterior to the engrailed domain allow shavenbaby expression and hence denticle formation. At the anterior side, converse conditions exist, since Wingless is present at high levels and the Egfr pathway is inactive. Therefore, it is proposed that polarization of Wingless transport by engrailed and hedgehog guarantees the naked fate anterior to the engrailed domain and the denticle fate posteriorly, and thus establishes the anteroposterior polarity of each segment (Sanson, 1999 and references).

It is conceivable that undetectable yet active Wingless is present in cells posterior to engrailed stripes. To demonstrate the absence of active Wingless there, a functional assay was used, based on the finding that wingless signaling represses rhomboid expression. From stage 11 onward, rhomboid is expressed in stripes just posterior to each engrailed domain, and this expression is abolished by continuous and uniform expression of Wingless. Later ectopic expression, induced at late stage 11, inhibits rhomboid transcription only in the midventral region, and if sibling embryos are left to develop, they make ectopic naked cuticle in the same region. Therefore, Wingless can repress rhomboid transcription in the same time window as it specifies naked cuticle. Wingless is not only sufficient for rhomboid repression, it is also necessary since wingless null mutants have an additional rhomboid stripe in each abdominal segment. The position of these extra stripes relative to landmarks in the CNS suggests that they form at the anterior of the domain of extinct engrailed expression, where wingless would normally be expressed. Thus, in the wild type, the presence of Wingless at the anterior of each engrailed stripe maintains the silence of rhomboid expression there. Significantly, rhomboid is expressed posterior to the engrailed domain of wild-type embryos. Therefore, active Wingless is not present in these cells, at least at late stage 11; if it were, rhomboid would not be expressed. These cells are located only two cell diameters posterior to the Wingless source (Sanson, 1999).

The asymmetric distribution of Wingless could be explained by decreased transport/stability either within the engrailed domain or at its posterior edge, where the segment boundary forms. To explore this, wingless was misexpressed directly in the engrailed domain (posterior to endogenous wingless) and it was determined whether the range of Wingless was shifted posteriorly. Wingless was expressed with the engrailed-Gal4 driver in otherwise wild-type embryos. The only effect on the cuticle pattern is the loss of row 1 denticles. Remarkably, no other denticles are lost. In particular, row 2 denticles are present even though they are adjacent to the Wingless-misexpressing cells. Thus wingless expressed at the anterior side of the presumptive segment boundary does not affect the fate of cells on the posterior side. To confirm this finding, rhomboid expression was used as an early molecular marker for the absence of Wingless. In the wild-type larva, rhomboid is expressed in the cells secreting rows 2-4. In en-Gal4/UAS-wg larvae, this expression is unchanged, indicating that the wingless pathway is not operative in the cells immediately posterior to the wingless-misexpressing cells. Thus, it appears that Wingless cannot cross the posterior edge of the engrailed domain. This was verified by looking directly at the distribution of Wingless protein in en-Gal4/UAS-wg embryos. In these embryos, Wingless is present within the domain of wingless misexpression, as expected. However, it is not detectable posterior to the engrailed-expressing cells. It is concluded that a barrier to Wingless protein movement exists at the presumptive segment boundary (Sanson, 1999).

The notion that Wingless movement is blocked at the forming segment boundary contrasts with an earlier proposal that Wingless spreads symmetrically. According to this view, posterior to its source, Wingless signaling is antagonized by active EGFR. The Egfr pathway is activated within and near the rhomboid stripe, which lies just posterior to the segment boundary. However, it is proposed that this segmental activation, which requires rhomboid, occurs after formation of the restrictions to Wingless movement. If wingless protein were present in the rhomboid cells at late stage 11, rhomboid expression would not be allowed there since wingless has been shown to repress rhomboid transcription. Subsequent establishment of rhomboid expression would further counteract activation of the Wingless pathway in prospective denticle belts (Sanson, 1999).

It is suggested that two mechanisms restrict posterior Wingless movement. The first restriction occurs within the engrailed domain and is unlikely to be under hedgehog control, since engrailed cells are not thought to respond to Hedgehog. Rather, engrailed could implement this restriction by controlling a gene involved in Wingless transport, sequestration, or stability. By contrast, the barrier at the posterior of the engrailed domain requires hedgehog signaling. Wingless produced ectopically in the engrailed domain of hedgehog mutants is allowed to invade posteriorly located cells and induce naked cuticle there. The finding that the same effects are seen in cubitus interruptus mutants indicates that the hedgehog signaling pathway is involved. The role of the hedgehog pathway is confirmed by "gain-of-function" experiments. Loss of patched results in overactivation of the hedgehog pathway and so does excessive hedgehog expression. Both situations reduce the range of Wingless in the anterior direction as if the spread of the protein were reduced. It is presumed that, in the wild type, a downstream Hedgehog target is upregulated at the posterior of each engrailed/hedgehog stripe and this would lead to Wingless destabilization or a block to transport there (Sanson, 1999).

The manner in which Hh molecules regulate a target cell remains poorly understood. In the Drosophila embryo, Hh is produced in identical stripes of cells in the posterior compartment of each segment. From these cells a Hh signal acts in both anterior and posterior directions. In the anterior cells, the target genes wingless and patched are activated whereas posterior cells respond to Hh by expressing rhomboid and patched. This study examines the role of the transcription factor Cubitus interruptus (Ci) in this process. So far, Ci has been thought to be the most downstream component of the Hh pathway, capable of activating all Hh functions. However, the study of a null ci allele indicates that it is actually not required for all Hh functions. Whereas Hh and Ci are both required for patched expression, the target genes wingless and rhomboid have unequal requirements for Hh and Ci activity. Hh is required for the maintenance of wingless expression before embryonic stage 11 whereas Ci is necessary only later during stage 11. For rhomboid expression Hh is required positively whereas Ci exhibits negative input. These results indicate that factors other than Ci are necessary for Hh target gene regulation. Evidence is presented that the zinc-finger protein Teashirt is one candidate for this activity. It is required positively for rhomboid expression and Teashirt and Ci act in a partially redundant manner before stage 11 to maintain wingless expression in the trunk (Gallet, 2000).

Loss of ci induces an expansion of rho expression instead of a reduction, as seen in a hh loss of function, showing that Ci is not involved in the activation of rho expression. The fact that rho disappears in tsh mutant embryos strongly suggests that the Tsh zinc-finger protein regulates rho expression or is at least necessary for instructing cells to respond to Hh for rho expression. Nevertheless, one has to explain why rho expression is expanded in ci94 . Loss of Cirep activity could be responsible for this effect. Indeed, overexpression of Cirep in a ci null background or analyses of the ciCe2 mutant, which ectopically expresses Cirep, reveals a repressive effect of Cirep on rho expression. Therefore, Cirep could be used as a gatekeeper in order to repress hh target genes tightly where they should not be expressed, and thus to overcome mis-regulation of key genes such as rho or wg. Nevertheless, these observations contradict previous analyses showing that Cirep is not required for correct embryogenesis, since loss of ci function is rescued by a ci transgene lacking the Ci75 repressor form of Ci. An alternative explanation can be gleaned from the fact that ci94 cuticle phenotypes resemble those lacking Wg activity during the cell specification stage. Because it has been shown that Wg exerts a repressive role on rho expression (since absence of Wg activity promotes ectopic expression of rho), rho expansion in ci94 could be an indirect consequence of the late disappearance of wg expression during stage 10-11 (Gallet, 2000).

Studies on the developing wing blade show that Ci transduces all Hh-delivered information. However, this study and others on the Hh pathway support the idea that Ci is not always involved in Hh signaling, showing that branchpoints are common for distinct Hh signaling steps for the following five reasons. (1) It has been shown that neither Ci nor Fused (Fu) are involved in the Hh-dependent formation of Bolwig's organ in Drosophila. (2) A Hh-responsive wg reporter gene with no Ci-binding sites does not require Ci activity for its regulation until stage 11. (3) Studies on the talpid 3 gene in chicken suggest that Gli proteins, the vertebrate homologues of Ci, regulate only a subset of Hh target genes, the others being regulated by an unidentified transcription factor. (4) A Sonic hedgehog response element on the COUP-TFII promoter binds to a factor distinct from Gli. (5) Hh signaling does not require Ci activity to regulate rho. Although the authors favor the idea that Tsh regulates rho expression directly in response to Hh signal the hypothesis that Tsh plays a more permissive role allowing Hh to regulate rho via another factor apart from Ci cannot be excluded (Gallet, 2000 and references therein).

In conclusion, Hh requires at least two different transcription factors during Drosophila embryogenesis to regulate its multiple target genes and to instruct cells with precise behaviors. The transcription factors may act independently (e.g. Ci for ptc; Tsh for rho), cooperatively (e.g. Ci and Tsh for wg maintenance during the cell specification phase) or redundantly (e.g. Ci and Tsh for wg maintenance earlier during the stabilization phase). The possibility that other transcription factors like gooseberry might be recruited for Hh signaling cannot be excluded, especially since denticle density is weaker in tsh;ci double mutants as compared with hh single mutants. Furthermore the dorsal phenotypes of the tsh;ci double mutants are weaker than those of hh. (1) wg transcripts are still present in dorsal patches in tsh;ci mutations whereas they are not present in hh embryos. (2) Dorsal cuticle is not as severely perturbed in tsh;ci larvae as compared with hh null ones (Gallet, 2000).

Convergent intercellular signals must be precisely integrated in order to elicit specific biological responses. During specification of muscle and cardiac progenitors from clusters of equivalent cells in the Drosophila embryonic mesoderm, the Ras/MAPK pathway -- activated by both epidermal and fibroblast growth factor receptors -- functions as an inductive cellular determination signal, while lateral inhibition mediated by Notch antagonizes this activity. A critical balance between these signals must be achieved to enable one cell of an equivalence group to segregate as a progenitor while its neighbors assume a nonprogenitor identity. Whether these opposing signals directly interact with each other has been investigated, and how they are integrated by the responding cells to specify their unique fates was been examined. Two distinct modes of lateral inhibition, one Notch based and a second based on the epidermal growth factor receptor antagonist Argos, are described that have complementary and reinforcing functions. Argos/Ras and Notch do not function independently; rather, several modes of cross-talk between these pathways have been uncovered. Ras induces Notch, its ligand Delta, and Argos. Delta and Argos then synergize to nonautonomously block a positive autoregulatory feedback loop that amplifies a fate-inducing Ras signal. This feedback loop is characterized by Ras-mediated upregulation of proximal components of both the epidermal and fibroblast growth factor receptor pathways. In turn, Notch activation in nonprogenitors induces its own expression and simultaneously suppresses both Delta and Argos levels, thereby reinforcing a unidirectional inhibitory response. These reciprocal interactions combine to generate the signal thresholds that are essential for proper specification of progenitors and nonprogenitors from groups of initially equivalent cells (Carmena, 2002).

This study involves the origin of two progenitors from a single cell cluster. The two progenitors are characterized by expression of the segmentation gene eve and are specified in a distinct temporal order in the Drosophila embryonic mesoderm. Progenitor 2 (P2) develops first; it originates from the preC2 cluster which develops into the C2 cluster and subsequently gives rise to a single P2 cell. P2 divides asymmetrically to give rise to two founder cells, one specific for a pair of persistently Eve-positive heart-associated or pericardial cells (EPCs) in every hemisegment and a second of previously undetermined identity. This second founder coexpresses Eve along with the gap gene Runt, with Eve levels rapidly fading but Runt persisting as development proceeds. By the time that Eve is evident in the EPCs, Runt labels a single somatic muscle, dorsal oblique muscle 2 (DO2). Runt is also detected in the muscle DO2 precursor during germband retraction (Carmena, 2002).

The second Eve progenitor, P15, which also has its origin in the preC2 cluster (which gives rise to a C15 cluster) forms later than P2 and divides asymmetrically to yield the founders of dorsal acute muscle 1 (DA1) and another cell whose fate cannot be followed since a specific, stably expressed marker for it is unavailable (Carmena, 2002).

To further substantiate the lineage relationships among these progenitors and founders, observations related to RTK signaling dependence of P2 and P15 specification were used: whereas P15 requires the activities of both Egfr and Htl, only Htl is involved in P2 formation. In this way, targeted mesodermal expression of a dominant negative form of Egfr strongly blocks formation of DA1 but not the EPCs. Also, consistent with DO2 and EPC founders being the progeny of P2, DO2 development, like that of the EPCs, is not affected by dominant negative Egfr. Additional support for the sibling relationship between the DO2 and EPC founders derived from the analysis of targeted expression of a dominant negative form of Htl. Under conditions in which early mesoderm migration is not perturbed, dominant negative Htl generates an incompletely penetrant phenotype in which different hemisegments lose derivatives of P2, P15, or both progenitors. With such partial inhibition of Htl activity, muscle DO2 and the EPCs are consistently either both present or both absent from any given hemisegment; in no cases did one of these cell types develop without the other, as expected for cells derived from a common progenitor. In contrast, muscle DA1 frequently forms in the absence of muscle DO2 and the EPCs, consistent with its derivation from an independent progenitor. Taken together, these data establish that the EPC and DO2 founders are sibling cells of the P2 division, whereas the other Eve-expressing muscle founder arises from a different progenitor (Carmena, 2002).

This model differs from one derived on the basis of clonal analysis in which it was proposed that the two Eve-positive mesodermal cell types originate from the same progenitor. This discrepancy may relate to the fact that muscles form by sequential cell fusions involving both founders and fusion-competent cells of potentially different parental cell origins, thereby confounding the interpretation of clonal analysis in which the cytoplasm of a single myotube is labeled by the lineage tracing marker (Carmena, 2002).

Autoregulation of a signal transduction cascade can cause either enhancement or attenuation of the transduced signal, depending on whether the feedback loop acts positively or negatively. Both types of feedback control occur during the Ras- and N-mediated specification of Eve mesodermal progenitors. Ras activation leads to increased expression of several proximal components of both the Fgfr and Egfr pathways that serve to amplify and/or prolong both fate-inducing RTK/Ras signals in the emerging Eve progenitors. A similar amplification of Egfr signaling occurs via induction of Rho during Drosophila oogenesis and mesothoracic bristle formation, and via upregulation of Egfr expression during C. elegans vulva development. The present analysis also uncovers a positive feedback mechanism for inductive Fgfr signaling, in this case via increased expression of not only the Htl receptor but also its specific signal transducer, Heartbroken (Hbr). Interestingly, the data suggest that the downstream components may respond to different thresholds of Ras activity since Rho exhibits a less robust response than either Htl or Hbr to Ras activation (Carmena, 2002).

Competitive cross-talk between Ras and N is manifest by the ability of the latter to block the expression of proximal components of the two RTK pathways—namely Htl/Hbr and Rho -- as well as to prevent the associated activation of MAPK. An antagonistic relationship between the RTK and N pathways is also revealed by the strong genetic interaction between Dl and Egfr, in agreement with previously reported genetic studies. Collectively, these results establish that the RTK and N pathways are not simply acting in parallel to exert opposing influences on progenitor specification; rather, N must be interfering with the generation and/or transmission of the inductive RTK signal. This effect could occur at multiple levels. The ability of activated N to at least partially block MAPK activation induced by constitutive Ras argues that N functions downstream of Ras. An additional direct effect of N on expression of Ras-responsive target genes cannot be excluded, particularly since Enhancer of split repressors are involved in the specification of progenitor cell fates. Such targets could include eve itself, or, given positive autoregulation of RTK signaling, one or more RTK pathway components (Carmena, 2002).

An early step in the development of the large mesothoracic bristles (macrochaetae) of Drosophila is the expression of the proneural genes of the achaete-scute complex (AS-C) in small groups of cells (proneural clusters) of the wing imaginal disc. This is followed by a much increased accumulation of AS-C proneural proteins in the cell that will give rise to the sensory organ, the SMC (sensory organ mother cell). This accumulation is driven by cis-regulatory sequences, SMC-specific enhancers, that permit self-stimulation of the achaete, scute and asense proneural genes. Negative interactions among the cells of the cluster, triggered by the proneural proteins and mediated by the Notch receptor (lateral inhibition), block this accumulation in most cluster cells, thereby limiting the number of SMCs. In addition, proneural proteins trigger positive interactions among cells of the cluster that are mediated by the Epidermal growth factor receptor (Egfr) and the Ras/Raf pathway. These interactions, which are termed 'lateral co-operation', are essential for macrochaetae SMC emergence. Activation of the Efgr/Ras pathway appears to promote proneural gene self-stimulation mediated by the SMC-specific enhancers. Excess Egfr signaling can overrule lateral inhibition and allow adjacent cells to become SMCs and sensory organs. Thus, the Egfr and Notch pathways act antagonistically in notum macrochaetae determination (Culí, 2001).

In the notum anlagen the expression of rho/ve occurs mainly in proneural clusters and this expression is dependent on ac-sc. Rho/ve facilitates the processing of Spitz, an activating ligand of Egfr. The soluble, active form of Spitz promotes ectopic sc expression and SMC emergence. Hence, these data suggest that, in proneural clusters, Ac-Sc promote expression of rho/ve, which by activating Spitz, would stimulate Egfr signaling in the cells of the cluster. (The Vein Egfr ligand probably does not specifically act in proneural clusters, because many of these lie outside of its expression domain). It is thus proposed that Egfr mediates a mutual positive signaling among cells of the proneural cluster, which promotes SMC emergence by probably reinforcing ac-sc expression. This positive signaling is called lateral cooperation. Evidently, this does not exclude an autocrine activation of the Egfr pathway in the cells that express AS-C proteins, but the lateral cooperation hypothesis is favored since it is well established in other systems that the Egfr pathway is used mainly for intercellular communication. This signaling should facilitate the acquisition of the SMC state by one or a few cells of a proneural cluster (Culí, 2001).

Egfr-mediated lateral cooperation should tend to activate the SMC-specific enhancers in many cells of the proneural clusters. This, however, is prevented by N signaling, which is activated by Ac and Sc in the cells of the cluster. This signaling, by means of the bHLH proteins of the E(spl)-C, blocks the ac-sc-ase self-stimulatory loop promoted by the SMC-specific enhancers. However, within a proneural cluster the cells of the proneural field accumulate greater amounts of Ac-Sc proteins. As it has been hypothesized that cells that signal the most are the least inhibited by their neighbors, eventually, a cell of the proneural field will be released from the inhibitory loop and its levels of E(spl)-C bHLH protein will become minimal. This cell will turn on the ac-sc-ase self-stimulation and become an SMC. The SMC signals maximally to its neighbors and prevents them from following the same fate (lateral inhibition). These results add to this scenario the requirement for Egfr-mediated signaling for one cell of the proneural field to turn on the ac-sc-ase self-stimulatory loops and become an SMC. According to this model, Ac-Sc activate both the N-and Egfr-mediated signaling pathways, with their SMC-suppressing and SMC-promoting abilities, respectively, and both signaling systems appear to act on the same SMC-specific enhancers. Since an excess signaling by the N or the Egfr pathway will either prevent SMC determination or promote emergence of ectopic SMCs, the respective levels of signaling should balance each other so that only one SMC is determined at a time from each proneural cluster. How is this balance accomplished? This is at present unclear. The large enhancement of rho/ve mRNA in proneural clusters under conditions of insufficient N signaling suggests that this pathway may prevent the Rho/Ve-promoted activation of Egfr from rising to excessively high levels. In contrast, the insensitivity of the levels of E(spl)-m8 protein to the overexpression of UAS-aos in proneural clusters suggests that the Egfr pathway does not affect N signaling. Antagonistic interactions between the N and the Egfr pathways are found in other developing systems, as in the wing preveins and in the reiterative recruitment, from a long-lived atonal proneural cluster, of the precursors of the 70-80 scolopidia of the femoral chordotonal organs. In this later case, Egfr signaling promotes commitment of neural precursors and the Dl-N interaction prevents too many cells from being committed (Culí, 2001).

The segment polarity gene lines (lin)was identified by Nusslein-Volhard because of its effects the dorsal epidermal pattern. Evidence is provided that Lin is involved in stage specific Wingless signaling activity; it acts in the cell receiving the Wg signal. In addition, Lin can localize to the nuclei of cells signaled by Wg-secreting cells. It is hypothesized that Lin interacts with nuclear Wg signal transducers and confers stage specificity to the pathway. Lin also localizes to the cytoplasm of cells receiving the Hedgehog signal, suggesting that Hh competes with Wg signaling by exporting Lin from the nucleus. The second Wg-dependent target gene is ve (veinless or rhomboid), which is expressed in a row of cells posteriorly adjacent to the En/Hh-expressing cells. This spatially restricted pattern is regulated in ventral epidermis by Wg signaling. Wg regulates ve expression dorsally as well. For example, if Wg function is inactivated at late stages, ve is ectopically expressed anterior to the En domain. Reciprocally, if the Wg pathway is broadly activated, ve expression is repressed. Thus, ve expression also provides a molecular readout for the Wg pathway. In lin mutant embryos, a second stripe of ve is induced anterior to the En/Hh cells in the dorsal epidermis. Thus, Lin and Wg function are similarly required to repress ve gene expression. It is concluded that Lin acts in concert with Wg in regulating target genes and consequently patterning the dorsal cell types (Hatini, 2000).

The initiation of mesoderm differentiation in the Drosophila embryo requires the gene products of twist and snail. In either mutant, the ventral cell invagination during gastrulation is blocked and no mesoderm-derived tissue is formed. One of the functions of Snail is to repress neuroectodermal genes and restrict their expressions to the lateral regions. The derepression of the neuroectodermal genes into the ventral region in snail mutants is a possible cause of defects in gastrulation and in mesoderm differentiation. To investigate such a possibility, a series of snail mutant alleles was analyzed. Different neuroectodermal genes respond differently in various snail mutant backgrounds. Due to the differential response of target genes, one of the mutant alleles, V2, which manifests reduced Snail function, also shows an intermediate phenotype. In V2 embryos, neuroectodermal genes, such as single-minded and rhomboid, are derepressed while ventral invagination proceeds normally. However, the differentiation of these invaginated cells into mesodermal lineage is disrupted. The results suggest that the establishment of mesodermal cell fate requires the proper restriction of neuroectodermal genes, while the ventral cell movement is independent of the expression patterns of these genes. The expression of some ventral genes disappear in snail mutants. Snail function is required for activation of genes such as dGATAb (serpent) and zfh-1. It is proposed that Snail may repress or activate another set of target genes, including folded gastruation, that are required specifically for gastrulation (Hemavathy, 1997).

Short gastrulation prevents Decapentaplegic from suppressing neurogenesis laterally in the blastoderm embryo. It is possible to exacerbate defects in sog mutants by increasing the level of DPP. The earliest neuroectodermal marker affected in sog mutants with a double dose of dpp is rhomboid, which is normally expressed in lateral stripes 8-10 cells wide in wild-type embryos but rapidly narrows to stripes 4-6 cells across in sog mutants with elevated DPP. Similarly l'sc expression is reduced in sog mutants with elevated DPP (Biehs, 1996).

The single-minded (sim) gene of Drosophila encodes a nuclear protein that plays a critical role in the development of the neurons, glia, and other nonneuronal cells that lie along the midline of the embryonic CNS. sim is required for midline expression of a group of genes including slit, Toll, rhomboid, engrailed, and a gene at 91F (Nambu, 1990).

Formation of the trachea occurs by the migration and fusion of clusters of ectodermal cells specified in each side of ten embryonic segments. Morphogenesis of the tracheal tree requires the activity of many genes, among them breathless (btl) and ventral veinless (vvl), whose mutations abolish tracheal cell migration. Activation of the btl receptor by branchless (bnl), its putative ligand, exerts an instructive role in the process of guiding tracheal cell migration. decapentaplegic determines vvl expression along the embryonic dorsoventral axis; expansion of dpp expression results in an increased recruitment of cells to express vvl. These cells are allocated in the expanded tracheal placodes, indicating that expansion of dpp expression causes a concomitant enlargement of the traceal placodes and of vvl expression. vvl is also required for the maintenance of btl expression during tracheal migration (Llimargas, 1997).

vvl is independently required for the specific expression in the tracheal cells of thick veins (tkv) and rhomboid (rho), two genes whose mutations disrupt only particular branches of the tracheal system. Expression in the tracheal cells of an activated form of tkv, the Decapentaplegic receptor, induces shifts in the migration of these cells, asserting the role of the dpp pathway in establishing the branching pattern of the tracheal tree. In addition, by ubiquitous expression of the btl and tkv genes in vvl mutants it is shown that both genes contribute to vvl function. These results indicate that through activation of its target genes, vvl makes the tracheal cells competent to further signaling and suggest that the btl transduction pathway could collaborate with other transduction pathways also regulated by vvl to specify the tracheal branching pattern (Llimargas, 1997).

The Drosophila tracheal system arises from clusters of ectodermal cells that invaginate and migrate to originate a network of epithelial tubes. Genetic analyses have identified several genes that are specifically expressed in the tracheal cells and are required for tracheal development. Among them, trachealess (trh) is able to induce ectopic tracheal pits and therefore it has been suggested that it would act as an inducer of tracheal cell fates; however, this capacity appears to be spatially restricted. The expression of the tracheal specific genes in the early steps of tracheal development and their crossinteractions have been examined. There is a set of primary genes including trh and ventral veinless (vvl) whose expression does not depend on any other tracheal gene and a set of downstream genes whose expression requires different combinations of the primary genes. The combined expression of primary genes is sufficient to induce some downstream genes but not others. The Dpp and EGF pathways are required for migration of certain branches of the tracheal system; competence of the tracheal cells to those signals depends on the specific tracheal expression in the tracheal cells of tkv and rho, respectively. Similarly to the btl pathway, rho expression in the tracheal cells depends both on trh and vvl function. However, while tracheal expression of tkv also depends on vvl, it appears to be independent of trh. The opposite appears to be the case for two other tracheal genes, tracheal defective (tdf) and pebbled (peb) [also known as hindsight (hnt)], which code for two putative transcription factors. Both genes appear to be targets of trh but they are present in the tracheal cells of vvl mutant embryos. Thus, some tracheal genes seem to be common targets of vvl and trh but others seem to depend only on one of them (Boube, 2000).

The genes araucan and caupolican code for two divergent homeodomain proteins that regulate transcription from the position-specific enhancers of ac-sc. Expression in the wing imaginal disc starts during the second larval instar at the presumptive notum region and is increased in two large areas of the presumptive lateral heminotum. From the mid-third instar, expression occurs at the presumptive distal tegula, the dorsal radium, proximal vein L1, veins L3 and L5, the allula, and the pleura. This distribution suggests that ARA and CAUP in fact establish the prepattern for proneural clusters in the wing, and interact with the position-specific enhancers to regulate ac-sc in the very precise pattern displayed by these proneural clusters. This distribution of ara and caup transcription also suggests that the two genes are involved in establishing the prepattern for rhomboid, thus interacting with its position-specific enhancers to establish transcription at the sites of future veins (Gomez-Skarmeta, 1996).

The transcription factor encoded by spalt major (salm) gene, which is expressed in the wing disc in a broad wedge centered over the decapentaplegic stripe, is one target of Dpp signaling. The anterior edge of the salm expression domain abuts a narrow stripe of rhomboid (rho)-expressing cells corresponding to the L2 longitudinal vein primordium. rhomboid is transcribed in a pattern that matches the future site of vein formation. hh mis-expression along the anterior wing margin induces a surrounding domain of salm expression, the anterior edge of which abuts a displaced rho L2 stripe. salm plays a key role in defining the position of the L2 vein, since loss of salm function in mosaic patches induces the formation of ectopic L2 branches, composed of salm- cells running along clone borders at positions where salm- cells confront salm+ cells. These data suggest that salm determines the position of the L2 vein primordium by activating rho expression in neighboring cells through a locally non-autonomous mechanism. rho then functions to initiate and maintain vein differentiation. The sal expression pattern is determined by dpp signaling: the spacing of the L2 and L5 rhomboid stripes is sensitive to the level of dpp since various forms of dpp misexpression generated discs with an elongated A/P axis and with the L2 and L5 rhomboid stripes shifted away from the A/P border. These data suggest that the positions of the L2 and L5 vein primordia may be determined by threshold responses to DPP produced at the A/P boundary. These data provide the final link connecting the formation of a linear adult structure to the establishment of a boundary by the maternal Bicoid morphogen gradient in the blastoderm embryo (Sturtevant, 1997).

It is believed that wing veins L3 and L4 do not respond to DPP signaling but instead L3 is determined directly by a threshold response to Hedgehog secreted across the A-P compartment boundary. It has been observed that clones of mutant patched cells in the middle of the anterior compartment are surrounded by an ectopic L3 vein which comprises wild-type cells. Similarly, loss-of-function clones of Protein kinase A, which functions like Ptc to repress ptc expression, are encircled by ectopic veins consisting of wild-type cells. Thus, cells with low levels of ptc may induce adjacent ptc+ cells to assume L3 fates. Since secreted HH is thought to be responsible for inactivating PTC, the position of the L3 primordium might be determined by a threshold response to HH diffusing from the posterior compartment (Sturtevant, 1997 and references).

The knirps and knirps-related genes organize the development of the second wing vein in Drosophila. The position of the L2 wing vein is determined by a chain of known developmental events, beginning with the primary subdivision of the wing imaginal disc into anterior versus posterior lineage compartments. The subdivision of body segments such as the wing primordium into anterior and posterior compartments, in turn, can be traced back to early A/P patterning in the blastoderm stage embryo. To summarize these events briefly:

  1. The posterior compartment fate is defined by expression of engrailed (en), which activates expression of the short-range Hedgehog (Hh) signal in posterior compartment cells and prevents posterior compartment cells from responding to Hh.
  2. Secreted Hh travels a short distance (6-8 cells) into the anterior compartment where it initiates a sequence of signaling events, culminating in the activation of several Hh target genes including decapentaplegic.
  3. Dpp synthesized in this narrow strip of cells travels significant distances in both the anterior and posterior directions to activate expression of Dpp target genes, such as the neighboring salm (spalt-major) and spalt-related (salr) genes, in a threshold-dependent fashion.
  4. Juxtaposition of salm expressing and salm non-expressing cells induces expression of the rhomboid (rho) gene in a stripe 1-2 cells wide, corresponding to the L2 vein primordium.
  5. rho then promotes differentiation of all longitudinal veins during late larval and early pupal development by potentiating signaling through the Egf-R/RAS pathway (Lunde, 1998 and references).

Evidence is presented that kni and knrl link A/P positional information in larval wing imaginal discs to morphogenesis of the second longitudinal wing vein (L2). kni and knrl are expressed in similar narrow stripes corresponding to the position of the L2 primordium. The kni and knrl L2 stripes abut the anterior border of the broad central expression domain of the Dpp target gene spalt major (salm). Evidence is provided that radius incompletus (ri), a well-known viable mutant lacking the L2 vein, is a regulatory mutant of the kni/knrl locus. In ri mutant wing discs, kni and knrl fail to be expressed in the L2 primordium. In addition, the positions of molecular breakpoints in the kni/knrl locus indicate that the ri function is provided by cis-acting sequences upstream of the kni transcription unit. Consistent with kni and knrl playing a role in L2 vein formation, kni and knrl are expressed in similar narrow stripes corresponding to the position of the L2 primordium. kni-expressing cells abut the anterior border of strong sal-lacZ expression and express little or no detectable lacZ. For convenience, these kni expressing cells are referred to as salm non-expressing cells. Consistent with the genetic evidence that ri is a regulatory mutant of the kni/knrl locus, the L2 stripes of kni and knrl expression are absent in ri mutant discs. However, outside the wing pouch of ri discs, kni and knrl are expressed normally. In support of the genetic evidence suggesting that ri is a cis-acting regulatory allele of the kni/knrl locus, ri function has been mapped to a region lying immediately upstream of the kni transcription unit (Lunde, 1998).

Knirps targets rhomboid, which is required for the formation of the L2 vein. ri function is required to initiate expression of the vein-promoting gene rho in the L2 primordium, but is not essential for rho expression in other vein stripes. As would be expected if the kni/knrl locus acted upstream of rho, initiation of kni expression in the L2 primordium precedes that of rho. Another early marker for the L2 vein primordium is down-regulation of the key intervein gene blistered (bs). In ri mutants, down-regulation of Bs in L2 is not observed. Consistent with the kni/knrl locus functioning upstream of both rho and Egf-receptor signaling, kni and knrl are expressed normally in rho ve; vn 1 double mutant wing discs. rho ve; vn 1 mutants, which lack rho expression in vein primordia and have reduced levels of the Egf-R ligand encoded by the vn gene, are devoid of veins. Rescue of ri mutants by a ubiquitously expressed kni transgene also suggests that kni controls rho expression (Lunde, 1998).

The adjacent knirps and knirps-related (knrl) genes encode functionally related zinc finger transcription factors that collaborate to initiate development of the second longitudinal wing vein (L2). kni and knrl are expressed in the third instar larval wing disc in a narrow stripe of cells just anterior to the broad central zone of cells expressing high levels of the related spalt genes. A 1.4 kb cis-acting enhancer element from the kni locus has been identified that faithfully directs gene expression in the L2 primordium. Three independent ri alleles have alterations mapping within the L2-enhancer element; two of these observed lesions eliminate the ability of the enhancer element to direct gene expression in the L2 primordium. The L2 enhancer can be subdivided into distinct activation and repression domains. The activation domain mediates the combined action of the general wing activator Scalloped and a putative locally provided factor, the activity of which is abrogated by a single nucleotide alteration in the ri53j mutant. Misexpression of genes in L2 that are normally expressed in veins other than L2 results in abnormal L2 development. These experiments provide a mechanistic basis for understanding how kni and knrl link AP patterning to morphogenesis of the L2 vein by orchestrating the expression of a selective subset of vein-promoting genes in the L2 primordium (Lunde, 2003).

Once activated along the anterior sal border, kni and knrl organize development of the L2 vein, in part by activating expression of the key vein-promoting gene rho and by suppressing expression of the intervein gene blistered (bs). The kni locus is highly selective in regulating downstream gene expression in the L2 primordium as revealed by the observation that several genes expressed in other veins, such as caup and ara, ac and sc, and Delta, are excluded from the L2 primordium. Thus, the kni locus links patterning to vein-specific morphogenesis by functioning downstream of sal and upstream of genes involved in vein versus intervein development (Lunde, 2003).

The results described in this study demonstrate definitively that ri mutations are regulatory alleles of the kni locus disrupting the function of a cis-regulatory enhancer element that drives gene expression in the L2 primordium of wing imaginal discs. A crucial line of evidence supporting this conclusion is that mutant versions of the L2 enhancer incorporating either the 252 bp deletion present in kniri[1] or the single base pair substitution present in kniri[53j] eliminate the ability of this element to direct gene expression in the L2 primordium. In addition, it is possible to completely rescue the vein-loss phenotype of kniri[1] by expressing either the UAS-kni or UAS-knrl transgenes with an L2-GAL4 driver. Consistent with activation of rho being one of the key effectors of kni/knrl function, it is also possible to rescue the L2 vein-loss phenotype of kniri[1] by expressing a UAS-rho transgene in L2, although rescue is less complete and penetrant than that observed with UAS-kni or UAS-knrl (Lunde, 2003).

The veins in the Drosophila wing have a characteristic width, regulated by the activity of the Notch pathway. The expression of the Notch-ligand Delta (Dl) is restricted to the developing veins, and coincides with places where Notch transcription is low. The regulation of Notch, Dl and E(spl) expression occurs at the transcriptional level, DL mRNA being detected in the vein and Notch in broad stripes that correspond to the interveins in the third instar discs. The expression of Dl is maintained in pupal wings 24 hours after puparium formation (APF) in dorso-ventral stripes 608 cells wide, with those cells at vein-intervein boundaries accumulating maximal levels of DL mRNA. In contrast, the expression of Notch evolves during pupal development; it is gradually lost from intervein territories during the first 12 hours APF, becoming restricted in pupal wings 24 hours APF to stripes of 2-3 cells wide localized at the vein-intervein boundaries. At this stage, the cells that accumulate high levels of Notch mRNA correspond to those in which Dl expression is maximal. This asymmetrical distribution of ligand and receptor leads to activation of Notch on both sides of each vein within a territory of Delta-expressing cells, and to the establishment of boundary cells that separate the vein from adjacent interveins (de Celis, 1997).

The modulation (upregulation) of Notch expression at the vein/intervein boundaries is independent of the establishment of veins per se. Expression of E(spl)mß (see the Enhancer of split complex) is severly reduced in the wing pouch of veinlet vein double mutants, demonstrating that Notch, which normally serves to activate E(spl)mß is not activated vein/intervein boundaries. There is also a failure to accumulate Notch in vein/intervein boundaries when Notch signaling is strongly reduced, suggesting that this late expression of Notch depends on Notch signaling (de Celis, 1997).

In the intervein cells, the expression of the Enhancer of split gene is activated and the transcription of the vein-promoting gene veinlet is repressed, thus restricting vein differentiation. Notch signaling represses veinlet expression, as hyper-activation of Notch signalling results in the complete repression of veinlet in the imaginal disc. Conversely, reductions in Notch signaling result in an increased number of veinlet-expression cells in vein territories. Expression of Delta depends on the previous specification of veins by Egfr activity. Ectopic expression of veinlet in pupal wings, which serves to enhance Egfr activity, leads to ectopic expression of Delta in similar regions (de Celis, 1997).

It is proposed that the establishment of vein thickness relies on a combination of mechanisms that include:

There is a genetic interaction between blistered (DSRF) and rhomboid suggesting that blistered restricts the expression of rhomboid to vein regions. In blistered mutants rhomboid expression becomes more intense and widens to include regions normally fated to become intervein regions. The increased area of rhomboid anticipates the final vein phenotype of blistered mutants. Mutant blistered wing phenotypes range from ectopic venation and a moderate frequency of localized blistering through very thick posterior veins and a high frequency of blisters to a complete loss of adhesion between the two wing surfaces resulting in ballooned wings. In rhomboid/blistered double mutants the wing defects associated with bs mutations completely are suppressed (Fristrom, 1994).

bHLH-PAS proteins represent a class of transcription factors involved in diverse biological activities. Previous experiments have demonstrated that the PAS domain confers target specificity. This suggests an association between the PAS domain and additional DNA-binding proteins, which is essential for the induction of specific target genes. A candidate for interaction with Trh is Drifter/Ventral veinless, a POU-domain protein. A dual requirement for Trh and Drifter has been identified for the autoregulation of Trh and Drifter expression. Furthermore, ectopic expression of both Trh and Dfr (but not each one alone) triggers trh autoregulation in several embryonic tissues. A direct interaction between Drifter and Trh proteins, mediated by the PAS domain of Trh and the POU domain of Drifter, has been demonstrated (Zelzer, 2000).

Rho (Rhomboid) functions as a regulator for processing the EGF receptor ligand Spitz, and is expressed a embryonic stage 9/10 in the midline glial cells, as well as in cells positioned at the center of the tracheal placodes. The parallel expression of rho in the tissues where Sim and Trh are functional, suggests that it may be a transcriptional target of these two bHLH-PAS proteins. In trh mutant embryos, expression of rho in the tracheal placodes is abolished. Similarly, in sim mutant embryos, expression of rho in the midline is eliminated. To determine if rho expression is regulated by direct binding of Sim and Trh, a 762 bp fragment of the rho 50 regulatory region was dissected: this is sufficient for midline and tracheal expression. The sequence of this fragment contains four sites with the Sim/Trh (ST) binding consensus. Similar sites have previously been shown by in vivo and in vitro analysis to represent the binding sites for Sim/ARNT or Trh/ARNT heterodimers. The 762 bp rho regulatory region was further dissected, and the capacity of smaller fragments to induce midline or tracheal expression in embryos was followed. The following conclusions were reached: Sim/Trh binding sites STc and STd are neither sufficient nor necessary for tracheal or midline expression. In contrast, Sim/Trh binding sites STa and STb are essential for midline and tracheal expression. Distinct cis elements appear to be required to promote midline vs. tracheal expression (Zelzer, 2000).

The specification of vein versus intervein fate in the Drosophila wing disc crucially depends on the restriction of Rho expression to the primordial veins. Rho enhances Egfr signaling, which generates elevated levels of activated Map kinase (MAPK) essential for proper vein development and the suppression of intervein fate in vein cells. In the absence of Egfr signaling, either in clones mutant for the Drosophila Egfr or in wings of double rhomboid;vein (rhove;vn1) flies, veins fail to develop and assume an intervein fate. A counterpart of rho is net, whose transcription is confined to intervein sectors of third instar wing discs. Absence of net activity in third instar wing discs results in derepression of rho in all intervein regions except sector C (between veins L3 and L4) and initiates the formation of ectopic veins. Conversely, in the absence of rho activity, net is ectopically expressed in vein primordia of wing discs, as well as in distal veins L3 to L5 of pupal wings where differentiation of veins is suppressed. Thus, while net is repressed in vein cells by high Egfr signaling dependent on Rho, Net protein suppresses vein fate in intervein cells by repressing rho transcription. Repression of net in veins by high levels of Rho-dependent Egfr signaling is crucial for vein development, since ectopic expression of net is able to repress rho transcription and suppress vein fate. However, Egfr signaling in the absence of Rho is sufficient to initiate normal vein development in the proximal and anterior portions of the wing. Consistent with this finding, repression of rho by ectopic Net prevents vein formation only in regions where it depends on Rho (Brentrup, 2000).

Ubiquitous expression of rho or activated Ras represses net transcription in the entire wing disc and promotes vein development throughout the wing. Such flies have tube-like wings composed mainly, if not exclusively, of vein cells. Since this phenotype is much stronger than that of net null mutants, it follows (1) that additional factors that repress rho must be present in intervein regions, and (2) that ectopic Rho is able to suppress intervein fate in regions where such a fate is independent of net expression, and hence that in these regions rho is able to repress intervein-promoting and vein-suppression genes different from net. The first conclusion is also supported by the notion that rho does not expand into the intervein region between L3 and L4 in net mutants. The simplest explanation for these observations is that rho expression is controlled by a set of separate silencers responding to different repressors in different, perhaps overlapping intervein regions and that Net acts as one of the repressors of rho (Brentrup, 2000).

Because of the necessity for the concomitant suppression of the alternative fate when vein or intervein fates are specified, a system evolved in which Rho determines vein development by repressing net and later bs, which in turn specify intervein fate by repressing vein development in intervein regions. Such a balanced system is intrinsically labile unless it is stabilized through feedback loops. Multiple feedback loops operate at all tiers of vein fate regulation, and tiers are closely linked and overlap in time, which further enhances the stability of the system because it generates redundancy. It is proposed that the functions of Net and Bs are partially redundant because they both repress rho in intervein regions during overlapping, though not identical, developmental periods. Thus, while Net represses rho in all intervein sectors of third instar wing discs except sector C, Bs begins to repress rho in these regions only in early prepupal wings. In view of this hypothesis, it might be less surprising that the wing phenotypeof net null mutants is much weaker than that resulting from ubiquitous expression of rho, which also represses net completely, but converts almost the entire wing into vein material. It is assumed that the lack of Net function in net minus wing discs is partially compensated by the activation of bs, whose product represses rho in most of the intervein regions during the prepupal and pupal stage. This assumption is consistent with the observations and with the earlier finding that bs null mutants exhibit a wing phenotype very similar to that resulting from ubiquitous expression of rho in the developing wing. The rhove -like phenotype obtained after ubiquitous expression of Net during wing development is largely explained by the ability of Net to repress rho. The partial redundancy of net and bs functions in wing discs is supported by experiments in which ubiquitous expression of Net is still able to suppress the strong ectopic vein formation phenotype of bs mutants (the phenotype is indistinguishable from that produced in a net1 mutant background). In addition, bs expression is reduced in distal portions of net1 wing discs and hence appears to depend partially on Net, a finding that is consistent with the observation that LacZ expression of a bs enhancer trap line is ectopically activated and enhanced after ectopic expression of Net in bs wing discs (Brentrup, 2000).

Wings of nemo mutants are rounder and shorter than wildtype, contain extra vein material and are held away from the body at a 45° angle (Choi, 1994). A genetic second-site modifier screen was carried out to identify genes that either participate in Notch signaling or modulate cross-talk between signal transduction pathways. adirondack (adk) mutations, now known to be alleles of nmo, dominantly modify the rough eye phenotype caused by expression of activated Notch under expression of the sevenless promoter. The extra vein phenotype seen in hypomorphic nmo mutant alleles (nmoadk) suggests that the gene product normally functions in the suppression of the vein fate. The patterning of wing veins has been well characterized and involves many genes at various stages of specification, refinement and maintenance of vein fates. The roles of the EGF receptor and Notch pathways in these processes are well-characterized. One of the earliest markers for presumptive vein cells is the rhomboid gene. rhomboid facilitates signaling via the EGF receptor and its expression is negatively regulated by Notch. To examine the development of the ectopic veins in nmoadk, pupal wings were dissected and the pattern of Rhomboid mRNA expression was determined. In nmoadk pupal wings ectopic rhomboid expression is seen in the regions corresponding to where ectopic veins are found in adult wings. These results suggest that nmo normally acts to inhibit rho expression (Verheyen, 2001).

Inductive patterning mechanisms often use negative regulators to coordinate the effects and efficiency of induction. During Spitz EGF-mediated neuronal induction in the Drosophila compound eye and chordotonal organs, Spitz causes activation of Ras signaling in the induced cells, resulting in the activation of Ets transcription factor Pointed P2. Developmental roles are described for a novel negative regulator of Ras signaling, EDL/MAE (Modulator of the activity of Ets), a protein with an Ets-specific Pointed domain but not an ETS DNA-binding domain. The loss of EDL/MAE function results in a reduced number of photoreceptor neurons and chordotonal organs, suggesting a positive role in the induction by Spitz EGF. However, EDL/MAE functions as an antagonist of Pointed P2, by binding to its Pointed domain and abolishing its transcriptional activation function. Furthermore, edl/mae appears to be specifically expressed in cells with inducing ability. This suggests that inducing cells, which can respond to Spitz they themselves produce, must somehow prevent activation of Pointed P2. Indeed hyperactivation of Pointed P2 in inducing cells interferes with their inducing ability, resulting in the reduction in inducing ability. It is proposed that EDL/MAE blocks autocrine activation of Pointed P2 so that inducing cells remain induction-competent. Inhibition of inducing ability by Pointed probably represents a novel negative feedback system that can prevent uncontrolled spread of induction of similar cell fates (Yamada, 2003).

The edl/mae gene was identified through enhancer trap lines that harbor P-element insertions at 55E. mae encodes a 177 amino acid polypeptide that contains a region similar to the Pointed domain found in many Ets proteins. In contrast to all other proteins that contain the Pointed domain, EDL/MAE lacks the conserved DNA-binding domain, the ETS domain. Because of the potential function of EDL/MAE in Ras/MAPK signaling, the expression pattern of mae was examined in two tissues where Ets proteins function as downstream targets of Ras/MAPK signaling. In the eye imaginal disc mae mRNA is expressed in clusters of cells in two rows in the morphogenetic furrow. Expression is seen in a small number of cells in each cluster, with a spacing roughly corresponding to that of the ommatidial clusters. To examine mae expression at the cellular level, an mae enhancer trap line maeJS was used that expresses lacZ in the eye imaginal disc. Expression of this mae-lacZ reporter initiates in R8 cells within the morphogenetic furrow, corresponding to the stage in which R8 induces R2 and R5. Subsequently, R2/R5, which act as the secondary source of induction, also initiate edl/mae-lacZ expression at lower levels. During the development of the embryonic chordotonal organs, mae mRNA is present in chordotonal organ precursor (COP) C1-C5, but was undetectable in C6-C8. As in the eye imaginal disc, mae expression is transient and disappears from the COPs before they started dividing. Thus, in both the ommatidium and the chordotonal organ, mae expression is detectable only in cells with inducing ability (Yamada, 2003).

Analysis of mae mutants reveals that in both the eye and chordotonal organ, the loss of mae reduces the efficiency of Spitz-mediated induction. In retinal sections of maeJV/Df(2R)P34 and maeJV/L19 animals, about 3% of ommatidia show loss of photoreceptor cells, of the R1-R6 and R7 photoreceptor subtype. The R8 cell, which most strongly expresses mae expression within the ommatidium, is always present, even in ommatidia where other photoreceptor cells are missing. A similar phenotype is seen in maeL19 mutant clones, which entirely lack mae function. This phenotype is almost completely rescued by an mae+ transgene. The requirement of mae is more pronounced when the level of the inducing signal is compromised. Star is a dosage-sensitive component of Spitz-mediated induction in the eye, and is required for the transport of Spitz EGF to the Golgi apparatus. In Star-/+ animals, 30% of ommatidia show a reduction in the number of photoreceptor neurons, with the average number of R1-R7 cells reduced per ommatidium of 0.39. When maeJV/L19 mutation is placed in the Star-/+ background, 65% of ommatidia lacked at least one neuron, with 1.71 photoreceptor cells missing per ommatidium on average. Similarly, the mae mutation enhances the reduction in the number of photoreceptor neurons in a hypomorphic allele of spitz. These synergistic effects of mae and Star/spitz suggest that mae participates in the induction of R1-R7 by Spitz EGF (Yamada, 2003).

Within the developmental contexts examined in this study, mae expression appears to be confined to cells with the ability to induce other cells using Spitz EGF. This suggests that Mae may have a role in regulating induction by Spitz. Secreted Spitz acts not only on the induced cells, but is also received by the inducing cells themselves. Although the molecular events leading to the activation of Pnt within the induced cells is well established, whether the same regulatory cascade operates within the inducing cells had not been studied. Hyperactivation of Pnt in inducing cells was found to have a deleterious effect on induction; in the embryo, COP C3 loses expression of rhomboid, a factor that is essential for the production of Spitz EGF. Although inducing cells are positioned so that they receive highest levels of Spitz EGF that they produce, they may possess a mechanism to prevent hyperactivation of Pnt. The phenotypes of the mae loss-of-function mutants and the effect of Pnt hyperactivation are similar in both ommatidial and chordotonal organ development. Mae is thus likely to be a part of the machinery that antagonizes PntP2 to prevent the negative effect of Pnt on induction in the inducing cells (Yamada, 2003).

During both ommatidial assembly and the development of the chordotonal system, Pnt promotes neuronal development in the induced cells. It is suggested that Pnt may also suppress inducing ability in such cells. This would create a negative feedback loop so that the cell, once induced, does not itself acquire inducing ability. Although such a mechanism would be effective in preventing uncontrolled spread of homeogenetic induction, the need for such regulatory system arises only if induced cells also have the opportunity to acquire inducing ability. This is indeed the case for R2/R5; these cells form via induction by R8, and then express rhomboid and become a secondary source of Spitz EGF. Other cells, such as R3/R4 could also potentially become inducers, because they reside within the proneural cluster prior to the onset of induction and have probably experience Atonal expression, which promotes rhomboid expression. The repressive effect of Pnt on rhomboid would thus be a mechanism to safeguard against the potential activation of rhomboid by Atonal within the proneural cluster. Pnt may cause this repression via activating expression of a repressor or by acting as a repressor itself (Yamada, 2003).

The inhibition of rhomboid expression is not the only way that Pnt negatively regulates induction. In the eye, a rhomboid paralog roughoid plays a critical role in generating mature Spitz EGF. It is possible that roughoid may also be regulated by Pnt to control induction. Furthermore, upon activation of Ras signaling, induced cells produce negative regulators of the Ras pathway, such as Sprouty, Argos and Kekkon, generating negative feedback loops. Because Argos is a secreted antagonist of Spitz EGF, its production by inducing cells could be detrimental for induction. The inhibition of Pnt function by Mae may also serve to reduce Argos production in the inducing cells, allowing efficient induction (Yamada, 2003).

Polycomb (PcG) and trithorax (trxG) group genes are chromatin regulators involved in the maintenance of developmental decisions. Although their function as transcriptional regulators of homeotic genes has been well documented, little is known about their effect on other target genes or their role in other developmental processes. The patterning of veins and interveins in the wing has been used as a model with which to understand the function of the trxG gene ash2 (absent, small or homeotic discs 2). ash2 is required to sustain the activation of the intervein-promoting genes net and blistered (bs) and to repress rhomboid (rho), a component of the EGF receptor (Egfr) pathway. Moreover, loss-of-function phenotypes of the Egfr pathway are suppressed by ash2 mutants, while gain-of-function phenotypes are enhanced. These results also show that ash2 acts as a repressor of the vein L2-organising gene knirps (kni), whose expression is upregulated throughout the whole wing imaginal disc in ash2 mutants and mitotic clones. Furthermore, ash2-mediated inhibition of kni is independent of spalt-major and spalt-related. Together, these experiments indicate that ash2 plays a role in two processes during wing development: (1) maintaining intervein cell fate, either by activation of intervein genes or inhibition of vein differentiation genes, and (2) keeping kni in an off state in tissues beyond the L2 vein. It is proposed that the Ash2 complex provides a molecular framework for a mechanism required to maintain cellular identities in the wing development (Angulo, 2004).

Loss of ash2 function causes differentiation of ectopic vein tissue, indicating that ash2 is required for intervein development, where it functions as an activator of the intervein-promoting genes net and bs, restricting rho expression to vein regions. In addition, the loss-of-function phenotypes of Egfr alleles are rescued in ash2 mutants, while the gain-of-function phenotypes are enhanced. Furthermore, rho mRNA exhibits an expanded expression pattern in ash2 mutant tissues. Thus, ash2 promotes the maintenance of intervein fate, either by activation of net and bs or by repression of the Egfr pathway. Since rho and bs/net expression is mutually exclusive, it cannot be determined whether the Ash2 complex interacts directly with one or all of them. However, since bs expression is inhibited by the loss-of-function of ash2 during larval and pupal stages, it can be proposed that ash2 acts as a long-term chromatin imprint of bs that is stable throughout development (Angulo, 2004).

Hairless induces cell death by downregulation of EGFR signalling activity

Overexpression of the Notch antagonist Hairless (H) during imaginal development in Drosophila is correlated with tissue loss and cell death. Together with the co-repressors Groucho (Gro) and C-terminal binding protein (CtBP), H assembles a repression complex on Notch target genes, thereby downregulating Notch signalling activity. This study investigated the mechanisms underlying H-mediated cell death in S2 cell culture and in vivo during imaginal development in Drosophila. First, the domains within the H protein that are required for apoptosis induction in cell culture were mapped. These include the binding sites for the co-repressors, both of which are essential for H-mediated cell death during fly development. Hence, the underlying cause of H-mediated apoptosis seems to be a transcriptional downregulation of Notch target genes involved in cell survival. In a search for potential targets, transcriptional downregulation of rho-lacZ and EGFR signalling output were noted. Moreover, the EGFR antagonists lozenge, klumpfuss and argos were all activated upon H overexpression. This result conforms to the proapoptotic activity of H, as these factors are known to be involved in apoptosis induction. Together, the results indicate that H induces apoptosis by downregulation of EGFR signalling activity. This highlights the importance of a coordinated interplay of Notch and EGFR signalling pathways for cell survival during Drosophila development (Protzer, 2008).

This work allows two important conclusions: that overexpression of H induces cell-autonomous apoptosis, and that H requires the co-repressors Gro and CtBP for its proapoptotic activity. It is known that H assembles a repression complex together with the two co-repressors, resulting in transcriptional downregulation of Notch target genes. Hence, the ability of H to induce cell death is most likely a consequence of the repression of Notch target genes that are involved in cell survival. It is noted, however, that not every cell that receives an overdose of H dies. One simple explanation for this observation is that the only cells that die are those in which the relevant Notch target genes are normally active, as these cells require a Notch signal for survival. As H results in a repression of Notch activity, these cells would be driven into cell death, whereas those cells that do not depend on higher Notch levels for survival would be resistant to an H overdose. How is this effect of H realised at the molecular level? So far, it has not been possible to narrow down the analyses towards one target gene, the repression of which by the H repressor complex induces apoptosis. The most straightforward idea, repression of the anti-apoptotic protein Diap1, is not supported by the data. Instead, it was found that EGFR signalling activity is downregulated as a consequence of the upregulation of several negative regulators of EGFR (Protzer, 2008).

The existence of a densely woven network of genetic interactions between the EGFR and Notch signalling pathways is well established. This intensive cross-talk harmonises many developmental processes, such as proliferation, differentiation, cell fate specification, morphogenesis and programmed cell death. Still, the molecular basis of this genetic interplay remains largely obscure. So far, few molecular intersections between the Notch and EGFR pathways have been revealed. For example, EGFR signalling causes phosphorylation of the co-repressor Gro, thereby negatively modulating the transcriptional outputs of Notch signalling via the Enhancer of split [E(spl)] genes. Conversely, a myc-Gro complex was shown to inhibit EGFR signalling during neural development in the Drosophila embryo. Although mutual antagonism is probably the most prominent relationship in EGFR-Notch interactions, in some developmental situations both pathways cooperate to potentiate each other's signalling activities. One such example with regard to cell survival has been described in the retina of rugose mutant flies, where cell type-specific cell death could be reversed by an increase in Notch or EGFR signalling activity, indicating that both pathways adopt an anti-apoptotic function in this developmental context. Also, R7 photoreceptor cell specification requires the combined input of both Notch and EGFR signals. Moreover, Notch defines the scope of rho expression in the Drosophila embryo, thereby activating the EGFR pathway required for early ectodermal patterning. Also, during the development of mouse embryonic fibroblast, the Notch receptor-processing γ-secretase presenilin acts as a positive regulator of ERK basal level activity (Protzer, 2008).

A significant decrease was observed in the levels of activated MAPK (diP-ERK), which provides a good assessment of EGFR pathway activation, upon induction of H. Activated MAPK directly phosphorylates two transcription factors, Aop (Yan) and Pointed (Pntp2). Phosphorylation inactivates Aop, which in the unmodified state, represses EGFR targets. At the same time, phosphorylation activates Pointed, which then causes EGFR target gene transcription. As H is a well-defined transcriptional repressor of Notch target genes, it is most unlikely that it impedes EGFR activity at the level of phosphorylation. Moreover, it is not thought that H acts at the level of transcriptional regulation of EGFR target genes, even though combinatorial and antagonistic activities of the nuclear effectors of the EGFR and Notch signalling pathways have been described during eye development. Instead, the hypothesis is favored that H represses the transcription of EGFR activators, or might indirectly provoke the activation of EGFR repressors that affect, for example, the production of EGFR ligands or signal transduction (Protzer, 2008).

Rho activity is required for a timely and spatially regulated release of EGFR ligands. Accordingly, the expression of rho is highly dynamic during Drosophila development, and precedes the appearance of EGFR-induced activated MAPK. Hence, downregulation of rho by H would eventually result in lower levels of activated MAPK (diP-Erk). In contrast to other components of the EGFR signalling pathway, ectopic expression of rho results in EGFR activation in a wide range of tissues, indicating that Rho is an essential and limiting factor. So far, transcriptional control is the only known means of rho regulation. The complex array of enhancers regulating rho expression reflects the dynamic pattern of EGFR activation throughout Drosophila development (Protzer, 2008).

Interestingly, a transcriptional repression of rho-lacZ was observed in H gain-of-function clones that was dependent on the co-repressors Gro and CtBP. This effect might very well be direct, because it was shown previously that rho transcription is regulated by Su(H) in the neuroectoderm as well as in the gut of the Drosophila embryo. As mentioned above, Notch signalling has also been shown to regulate rho expression in the embryonic ectoderm. Moreover, during egg development, a band of Notch activity establishes the boundary between the two dorsal appendage tube cell types, whereby Notch levels are high in rho-expressing cells. In accordance with this, potential Su(H)-binding sites are present in the regulatory regions of rho1 and rho3, making a direct regulation of rho during eye development via the Notch-Su(H)-H complex very likely. It is noted, however, that the downregulation of rho-lacZ and of activated MAPK were focussed at the morphogenetic furrow, where primary photoreceptor cells are specified and ommatidia are founded. Regulation of rho by H would then be expected to interfere with photoreceptor formation rather than with cell survival, which is in agreement with the disturbed cellular architecture of H gain-of-function flies (Protzer, 2008).

Most interestingly, upon H overexpression, ectopic induction of lz, klu and aos was observed. All three genes are known to be involved in cell death induction during pupal eye development. There it was shown that the Runx protein Lz binds to the regulatory regions of klu and aos, resulting in the direct transcriptional activation of these target genes. Therefore, one might speculate that H executes its effect on klu and aos activity via the activation of lz. Moreover, as klu and aos are well-known inhibitors of EGFR signalling activity, this in itself suggests that H impedes EGFR signalling activity via these factors. This interpretation helps to explain why aos expression is induced in H gain-of-function clones, although it is well known that aos is triggered by EGFR signalling, thereby forming an inhibitory loop that acts on EGFR activity. The high levels of Lz still activate aos in H gain-of-function clones, keeping activity of the EGFR pathway low. Alternatively, aos and klu levels might be increased as a consequence of the downregulation, by H, of an as yet unknown repressor. Since H behaves as a kind of 'multi-adaptor protein', which not only recruits the transcriptional silencers Gro and CtBP to Notch targets but also binds other proteins such as Pros26.4, it is also possible that H interacts with positive regulators of lz, klu and aos (Protzer, 2008).

However, a model is favored whereby H influences EGFR signalling activity on two levels. On the one hand, through transcriptional repression of rho, H causes a loss of EGFR signalling output that interferes with cell specification. On the other hand, by interfering with their repressor(s), H relieves the restriction on lz, klu and aos expression, causing their accumulation. In consequence, the survival/death balance is tipped towards apoptosis in those cells that are susceptible to the effects of a lowered EGFR signal. Those cells that do not depend on high Notch and EGFR activity levels for survival would be resistant to an H overdose (Protzer, 2008).

Finally, one can envisage that a downregulation of Notch and EGFR signalling activities, resulting from the overexpression of H, might leave a cell in a state of 'uncertainty' that does not allow any further differentiation towards a certain cell type, but leaves the cell vulnerable to the apoptotic programme (Protzer, 2008).

Coordinated sequential action of EGFR and Notch signaling pathways regulates proneural wave progression in the Drosophila optic lobe.

During neurogenesis in the medulla of the Drosophila optic lobe, neuroepithelial cells are programmed to differentiate into neuroblasts at the medial edge of the developing optic lobe. The wave of differentiation progresses synchronously in a row of cells from medial to the lateral regions of the optic lobe, sweeping across the entire neuroepithelial sheet; it is preceded by the transient expression of the proneural gene lethal of scute [l(1)sc] and is thus called the proneural wave. This study found that the epidermal growth factor receptor (EGFR) signaling pathway promotes proneural wave progression. EGFR signaling is activated in neuroepithelial cells and induces l(1)sc expression. EGFR activation is regulated by transient expression of Rhomboid (Rho), which is required for the maturation of the EGF ligand Spitz. Rho expression is also regulated by the EGFR signal. The transient and spatially restricted expression of Rho generates sequential activation of EGFR signaling and assures the directional progression of the differentiation wave. This study also provides new insights into the role of Notch signaling. Expression of the Notch ligand Delta is induced by EGFR, and Notch signaling prolongs the proneural state. Notch signaling activity is downregulated by its own feedback mechanism that permits cells at proneural states to subsequently develop into neuroblasts. Thus, coordinated sequential action of the EGFR and Notch signaling pathways causes the proneural wave to progress and induce neuroblast formation in a precisely ordered manner (Yasugi, 2010).

Loss of EGFR function in progenitor cells caused failure of L(1)sc expression and differentiation into neuroblasts (see A model of progression of the proneural wave). In addition, elevated EGFR signaling resulted in faster proneural wave progression and induced earlier neuroblast differentiation. The activation of the EGFR signal is regulated by a transient expression of Rho, which cleaves membrane-associated Spi to generate secreted active Spi. This study also demonstrated that Rho expression itself depends on EGFR function, and thus the sequential induction of the EGFR signal progresses the proneural wave. Clones of cells mutant for pnt were not recovered unless Minute was employed, suggesting that the EGFR pathway is required for the proliferation of neuroepithelial cells. However, the progression of the proneural wave is not regulated by the proliferation rate per se (Yasugi, 2010).

The function of the Notch signaling pathway in neurogenesis is known as the lateral inhibition. A revision of this notion has recently been proposed for mouse neurogenesis, in which levels of the Notch signal oscillate in neural progenitor cells during early stages of embryogenesis, and thus no cell maintains a constant level of the signal. The oscillation depends mainly on a short lifetime and negative-feedback regulation of the Notch effecter protein Hes1, a homolog of Drosophila E(spl). This prevents precocious neuronal fate determination. The biggest difficulty in analysis of Notch signaling is the random distribution of different stages of cells in the developing ventricular zone, which is thus called a salt-and-pepper pattern. In medulla neurogenesis, however, cell differentiation is well organized spatiotemporally and the developmental process of medulla neurons can be viewed as a medial-lateral array of progressively aged cells across the optic lobe. Such features allowed the functions of Notch to be precisely analyzed. Cells are classified into four types according to their developmental stages: neuroepithelial cells expressing PatJ, neuronal progenitor I expressing a low level of Dpn, neuronal progenitor II expressing L(1)sc and neuroblasts expressing high levels of Dpn. The Notch signal is activated in neuronal progenitor I and II. The EGFR signal turns on in the neuronal progenitor II stage and progresses the stage by activating L(1)sc expression. Cells become neuroblasts when the Notch and EGFR signals are shut off. Cells stay as neuronal progenitor I when Notch signal alone is activated, whereas cells stay as neuronal progenitor II when the Notch signal is activated in conjunction with the EGFR signal. Although the Notch signal is once activated, it must be turned off to let cells differentiate into neuroblasts. In neuronal progenitor II, E(spl)-C expression is induced by Notch signaling, and the increased E(spl)-C next downregulates Dl expression and subsequent activation of the Notch signal (Yasugi, 2010).

What does Notch do in medulla neurogenesis? It is infered that the Notch signal sustains cell fates, whereas the EGFR signal progresses the transitions of cell fate. This was well documented when a constitutively active form of each signal component was induced. EGFR, or its downstream Ras, induces expression of L(1)sc but does not fix its state, even though the constitutively active form is employed. At the same time, a constitutively active Notch sustains cell fates in a cell-autonomous manner. Constitutively active N receptors, by contrast, autonomously determine cell fates depending on the context: cells become neuronal progenitor I in the absence of EGFR and neuronal progenitor II in the presence of EGFR. The precocious neurogenesis caused by the impairment of Notch signaling suggests that Notch keeps cells in the progenitor state for a certain length of time in order to allow neuroepithelial cells to grow into a sufficient population. In the prospective spinal cord of chick embryo (Hammerle, 2007), the development from neural stem cells to neurons progresses rostrocaudally, during which the transition from proliferating progenitors to neurogenic progenitors is regulated by Notch signaling (Yasugi, 2010).

Although Notch plays a pivotal role in determining cell fate between neural and non-neural cells, the function may be context dependent and can be classified into three categories. (1) Classical lateral inhibition is seen in CNS formation in embryogenesis and SOP formation in Drosophila. Cells that once expressed a higher level of the Notch ligand maintain their cell states and become neuroblasts. (2) Oscillatory activations are found in early development of the mouse brain (Shimojo, 2008). Progenitor cells are not destined to either cell types. (3) An association with the proneural wave found in Drosophila medulla neurogenesis as is described in this study. The Notch signal is transiently activated only once and then shuts off in a synchronized manner. The notable difference in the outcome is the ratio of neural to non-neural cells; a small number of cells from the entire population become neuroblasts or neural stem cells in the former cases (1 and 2), whereas most of the cells become neuroblasts in the latter case (3). The differences between (1) and (2) can be ascribed at least in part to the duration of development. Hes1 expression has been shown to oscillate within a period of 2 hours in the mouse, whereas in Drosophila embryogenesis, selection of neuroblasts from neuroectodermal cells takes place within a few hours. Thus, even if Drosophila E(spl) has a half-life time equivalent to Hes1, the selection process during embryogenesis probably finishes within a cycle of the oscillation. The process of medulla neuroblast formation continues for more than 1 day, but Notch signaling is activated for a much shorter period in any given cell. This raises the possibility that E(spl)/Hes1 may have a similarly short half-life but outcome would depend on the developmental context (Yasugi, 2010).

The functions of EGFR and Notch described in this study resemble their roles in SOP formation of adult chordotonal organ development; the EGFR signal provides an inductive cue, whereas the Notch signal prevents premature SOP formation. In addition, restricted expression of rho and activation of the EGFR signal assure reiterative SOP commitment. Several neuroblasts are also sequentially differentiated from epidermal cells in adult chordotonal organs (Yasugi, 2010).

Unpaired, a ligand of the JAK/STAT pathway is expressed in lateral neuroepithelial cells and shapes an activity gradient that is higher in lateral and lower in the medial neuroepithelium. The JAK/STAT signal acts as a negative regulator of the progression of the proneural wave (Yasugi, 2008). This report has shown that activation of both EGFR and Notch signaling pathways depends on the activity of the JAK/STAT signal. The JAK/STAT signal probably acts upstream of EGFR and Notch signals in a non-autonomous fashion. These three signals coordinate and precisely regulate the formation of neuroblasts (Yasugi, 2010).

The Elongin complex antagonizes the chromatin factor Corto for Vein versus intervein cell identity in Drosophila wings

Drosophila wings mainly consist of two cell types, vein and intervein cells. Acquisition of either fate depends on specific expression of genes that are controlled by several signaling pathways. The nuclear mechanisms that translate signaling into regulation of gene expression are not completely understood, but they involve chromatin factors from the Trithorax (TrxG) and Enhancers of Trithorax and Polycomb (ETP) families. One of these is the ETP Corto that participates in intervein fate through interaction with the Drosophila EGF Receptor -- MAP kinase ERK pathway. Precise mechanisms and molecular targets of Corto in this process are not known. This study shows that Corto interacts with the Elongin transcription elongation complex. This complex, that consists of three subunits (Elongin A, B, C), increases RNA polymerase II elongation rate in vitro by suppressing transient pausing. Analysis of phenotypes induced by EloA, B, or C deregulation as well as genetic interactions suggest that the Elongin complex might participate in vein vs intervein specification, and antagonizes corto as well as several TrxG genes in this process. Chromatin immunoprecipitation experiments indicate that Elongin C and Corto bind the vein-promoting gene rhomboid in wing imaginal discs. It is proposed that Corto and the Elongin complex participate together in vein vs intervein fate, possibly through tissue-specific transcriptional regulation of rhomboid (Rougeot, 2013).

In Drosophila as in mammals, the three Elongin proteins Elo A, B, and C are mainly nuclear and interact two by two. EloC/B and EloC/A interactions may be direct, as they were observed without cross-linking treatment. By contrast, EloA/B interaction is more labile and may thus be indirect. It is possible that Drosophila EloC mediates the interaction between EloA and EloB, as previously shown in mammals. This study also showed that the ETP Corto interacts with all three Elo proteins, suggesting that Corto interacts with the Elongin complex. Hence, Corto and the Elongin Complex could share transcriptional targets. Several studies have shown that EloC binds its partners through a degenerate BC box motif, defined as (L,M)XXX(C,S)XXX(Í). Two putative BC boxes (aa 357-365 and aa 542-550) are present in the C-terminal part of Corto. However, deletion of these sequences did not impair co-immunoprecipitation between Corto and EloC, suggesting that these two proteins interact through another unidentified sequence (Rougeot, 2013).

This study presents the first characterization of lines allowing deregulation of EloB or EloC expression. EloB or EloC loss-of-function mutations induce early lethality (before the third larval instar), demonstrating that EloB and EloC, like EloA (Gerber, 2004), are essential proteins. Clonal and tissue-specific analyses of EloC mutant cells reveal that EloC is critically required all through wing development. By contrast, RNAi-mediated EloA down-regulation only induced lethality during the pupal stage (Gerber, 2004), indicating either a less efficient reduction of EloA mRNA or a longer perdurance of maternal EloA. Alternatively, requirement of EloB and EloC in other complexes, such as an E3 ubiquitin ligase complex, might explain this difference (Rougeot, 2013).

EloB/C loss-of-function as well as EloA over-expression induced wing phenotypes, mostly vein phenotypes. Interestingly, these loss-of-function and over-expression phenotypes are opposite (i.e truncated L5 vein for loss-of-function, ectopic veins for over-expression). Furthermore, whereas EloA over-expression induced ectopic veins, no phenotype was observed when over-expressing EloB and EloC. This result suggests that the amount of catalytic subunit EloA might be critical for Elongin complex function. In mammals, EloA is indeed the limiting component of the Elongin complex, EloB and EloC being in large excess (100 to 1000-fold more abundant than EloA). Curiously, a previous study reported that mitotic clones for a deficiency that uncovers EloA, produced ectopic wing veins. As this deletion uncovers more than 10 genes that may influence vein formation, the hypothesis is favored, in agreement with all data presented above, that EloA loss- of-function leads to loss of vein tissue. Alternatively, EloB and EloC, which also belong to ubiquitin ligase complexes, might modulate vein vs intervein cell fate in this context (Rougeot, 2013).

Altogether, the observations suggest that the Elongin A, B, C subunits promote vein cell identity. On the opposite, Corto maintains intervein cell identity, possibly via interaction with TrxG complexes. As Corto and EloC co-localize at a few sites on polytene chromosomes, they might have common transcriptional targets. A balance between Corto and the Elongin complex might fine-tune transcription of such genes (Rougeot, 2013).

In corto mutants, previous study has shown that ectopic veins perfectly match with ectopic expression of rho, the first vein-promoting gene to be expressed (Mouchel-Vielh, 2011). As Elo gene mutations counteract corto mutations during formation of ectopic veins, it is proposed that rho could be a common target of Corto and the Elongin complex in intervein cells. In agreement with this hypothesis, immunoprecipitation using chromatin from late third instar wing imaginal discs, that can be assimilated to chromatin of intervein cells, revealed the presence of both Corto and EloC on rho. Two independent genome-wide studies on whole embryos and embryonic S2 cells have shown that poised RNA-PolII binds the rho promoter, suggesting that rho expression is controlled by 'pause and release' of the transcriptional machinery. Interestingly, this studu found that Corto is slightly enriched just after the rho TSS, a position usually occupied by paused RNA-PolII. Corto shares many sites on polytene chromosomes with paused RNA-PolII-S5p, suggesting that it is involved in transcriptional pausing. On the other hand, this study found that EloC co-localizes with H3K36me3, that characterizes transcriptional elongation, and the Elongin complex was shown to suppress transient RNA-PolII pausing. Hence, in future intervein cells, Corto and the Elongin complex could apply opposite forces on the transcriptional machinery at the rho promoter. Corto would block rho transcription whereas the Elongin complex would be ready to accompany rho elongation if release should occur. In future vein cells on the other hand, the Elongin complex could actively participate in rho transcriptional elongation, since loss of function mutants for EloB and EloC exhibit loss of vein tissue. In these cells, rho expression would be independent of Corto, since corto mutants never present truncated veins (Rougeot, 2013).

The results suggest that the Elongin complex might participate in determination of vein and intervein cell identity during wing development. It is proposed that this complex might interact with the ETP Corto at certain target genes and fine-tune their transcription in a cell-type specific manner. One of these targets could be the vein-promoting gene rho. In intervein cells, binding of Corto to the Elongin complex could prevent transcription of rho. Corto could also recruit other chromatin factors, such as the BAP chromatin-remodeling complex that was previously shown to inhibit rho expression in intervein cells. By contrast, in vein cells, the Elongin complex could participate in rho transcriptional elongation independently of Corto (Rougeot, 2013).

Continued: Rhomboid Transcriptional regulation part 3/3 | back to part 1/3

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

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