Promoter Structure

Most cis-acting regulatory sequences of Delta lie within 6.6 kb immeditely upstream of the Delta transcription start site. A quantitiative enhancer of transcription is found in the Delta first intron. Sequences responsible for expression in the anterior cephalic domain are found from -6.6 to -3.3kb and -180 to +674 kb, and for the posterior expression domain and the thoracic abdominal domain from -4.3 to -3.3 kb, and -6.6 to -4.3 kb respectively. Regions that give a pair rule stripe pattern and a neuroectodermal expression pattern have also been identified (Haenlin, 1994).

Transcriptional Regulation

Transcription of Delta in the neuroectoderm is regulated by genes of the achaete-scute complex. Lethal of scute is expressed in the same temporal and spatial pattern as Delta. Achaete, Scute and Lethal of scute (but not Asense) activate Delta transcription in the neuroectoderm either directly or indirectly. Lethal of scute is sufficient to activate Delta in imaginal discs (Hinz, 1994). Suppressor of Hairless exhibits allele specific interactions with Delta, indicating that the Notch pathway may regulate Delta transcription by means of Suppressor of Hairless (Fortini, 1994).

The maternal Dorsal nuclear gradient initiates the differentiation of the mesoderm, neurogenic ectoderm and dorsal ectoderm in the precellular Drosophila embryo. Each tissue is subsequently subdivided into multiple cell types during gastrulation. This study investigates the formation of the mesectoderm within the ventral-most region of the neurogenic ectoderm. Previous studies suggest that the Dorsal gradient works in concert with Notch signaling to specify the mesectoderm through the activation of the regulatory gene sim within single lines of cells that straddle the presumptive mesoderm. This model was confirmed by misexpressing a constitutively activated form of the Notch receptor, NotchIC, in transgenic embryos using the eve stripe2 enhancer. The NotchIC stripe induces ectopic expression of sim in the neurogenic ectoderm where there are low levels of the Dorsal gradient. sim is not activated in the ventral mesoderm, due to inhibition by the localized zinc-finger Snail repressor, which is selectively expressed in the ventral mesoderm. Additional studies suggest that the Snail repressor can also stimulate Notch signaling. A stripe2-snail transgene appears to induce Notch signaling in 'naïve' embryos that contain low uniform levels of Dorsal. It is suggested that these dual activities of Snail -- repression of Notch target genes and stimulation of Notch signaling -- help define precise lines of sim expression within the neurogenic ectoderm. It is proposed that Snail functions as a gradient repressor to restrict Notch signaling. In precellular embryos, the initial snail expression pattern is broad and extends into the future mesectoderm. During cellularization, the pattern is refined and snail expression is lost in the mesectoderm and restricted to the mesoderm. The early, broad snail pattern might create a broad domain of potential Notch signaling by repressing components of the Notch pathway, such as Delta and lethal of scute. After cellularization, Notch signaling is blocked in the presumptive mesoderm by sustained, high levels of the Snail repressor. However, Notch can be activated in the mesectoderm because of the loss of Notch inhibitors repressed by transient expression of the Snail repressor. According to this model, the dynamic snail expression pattern determines both the timing and limits of Notch signaling (Cowden, 2002).

Enhancer of split complex genes regulate Delta by acting through achaete-scute complex genes. Mutation of E(spl)-C genes or groucho, like Notch or Delta mutants cause an overproduction of sensory organs precursors at the expense of epidermis. The mutant cells behave autonomously suggesting that the corresponding gene products are required for reception of the inhibitory signals. Epistasis experiments place both E(spl)-C genes and and groucho downstream of Notch and upstream of achaete and scute, consistent with the idea that they are part of the Notch signaling cascade. Like mutant Notch cells. Cells mutant for E(spl)-C genes or groucho inhibit neighouring wild-type cells destined for neural fate, thus causing them to adopt the epidermal fate. This inhibition requires the genes of the achaete-scute complex, which would be in a more active state in cells mutant for E(spl)-C genes or groucho. This active state would elevate Delta transcription, repressing the neural fate of neighboring cells. Thus there is a regulatory loop between Notch and Delta that is under the transcriptional control of the E(SPL)-C and AS-C genes (Heitzler, 1996).

Regulation of Delta in the wing

Notch function is required at the dorsoventral boundary of the developing Drosophila wing for its normal growth and patterning. Clones of cells expressing either Notch or its ligands Delta and Serrate in the wing mimic Notch activation at the dorsoventral boundary, producing non-autonomous effects on proliferation and activating expression of the target genes E(spl), wingless and cut. The analysis of these clones reveals several mechanisms important for maintaining and delimiting Notch function at the dorsoventral boundary:

Thus the combined effects of Notch and its target genes cut and wingless regulate the expression of Notch ligands, which restricts Notch activity to the dorsoventral boundary (de Celis, 1997c).

During vein differentiation dpp is expressed in the pupal veins under the control of genes that establish vein territories in the imaginal disc. Both dpp and thick veins are differentially expressed in vein territories during pupal development. dpp and tkv regulate one another by a feedback mechanism in which Tkv activity represses dpp expression. Dpp, acting through its receptor Thick veins, activates vein differentiation and restricts expression of both veinlet and the Notch-ligand Delta to the developing veins. Once Dpp is established in the veins, local activation of Tkv in these cells is required both for the maintenance of veinlet and Delta expression and for the veins to differentiate. In dpp mutants, the vein thickening observed in Notch mutants is elimated. Conversely, Notch gain-of-function alleles that lead to the truncation of veins results in very pronounced vein loss in combination with both dpp and tkv mutants. In dpp mutants, Delta and E(spl)mß, which normally takes place in vein territories, is lost. In summary, genetic combinations between mutations that increase or reduce Notch, veinlet and dpp activities suggest that the maintenance of the vein differentiation state during pupal development involves cross-regulatory interactions between these pathways (de Celis, 1997a).

The veins in the Drosophila wing have a characteristic width; this is 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 lower. 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, 1997b).

The modulation (upregulation) of Notch expression at the vein/intervein boundaries is independent of the establishment of veins per se. Expression of E(spl)mbeta is severly reduced in the wing pouch of veinlet vein double mutants, demonstrating that Notch, which normally serves to activate E(spl)mbeta 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, 1997b).

In the intervein cells, the expression of the Enhancer of split gene mbeta 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, 1997b).

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

Induction of Senseless (Sens) expression using the dpp-GAL4 driver alters Delta expression. The domain that normally gives rise to the third wing vein, is altered in Sens-overexpressing discs. Overexpression of Sens induces Delta expression ectopically in the dpp domain, broadening and intensifying the endogenous Delta domain. In addition, a consistent reduction of expression in the fourth wing vein domain is observed. This ectopic Delta expression is likely to be mediated by Scute/Asense overexpression (Nolo, 2000 and references therein).

To determine the relationship between Sens expression and the proteins of the Enhancer of Split complex, wild-type discs were stained for both proteins. There is little overlap between the two nuclear proteins. Cells that express Sens are intermingled with E(spl) expressing cells, but the majority of cells that express Sens do not express E(spl). Similar observations were also made with E(spl)m8-lacZ and with E(spl)m4-lacZ. These data indicate that Sens expression in cells fated to develop into SOPs is concomitant with the presence of E(spl) proteins, but that elevation of Sens expression and cell enlargement during SOP specification accompanies a rapid removal of the E(spl) protein. These data are also in agreement with the proposition that E(spl) is a negative regulator of proneural gene expression and that its downregulation permits SOP development (Nolo, 2000 and references therein).

Ectopic expression of Sens may not only activate the proneural genes and Delta but may recreate an ectopic proneural field. Expression of several E(spl) proteins depends on the presence of the proneural genes. Therefore Sens was overexpressed using the dpp-GAL4 driver in E(spl)m8-lacZ and E(spl)m4-lacZ imaginal discs. Wild-type discs contain proneural clusters that express cytoplasmic ßgalactosidase [E(spl)] in which few cells are Sens positive. Overexpression of Sens causes a strong induction of ßgalactosidase staining associated with E(spl)m4-lacZ and E(spl)m8-lacZ. This induction is not restricted to cells in which Sens is expressed but can be detected in adjacent cells as well. This indicates that Sens can induce in a cell-nonautonomous fashion E(spl) expression, probably by activating Delta expression. A more detailed cellular analysis shows that when Sens expression is elevated in a particular cell, ßgalactosidase levels are consistently low or absent. It is inferred that ectopic Sens leads to expression of the essential components required to establish a proneural domain in some areas of the wing discs. This ability is most likely mediated by its ability to activate the proneural genes. The wing hinge region is, however, refractory to induction of Scute, Delta, and E(spl) upon overexpression of Sens (Nolo, 2000).

During the development of the Drosophila wing, the activity of the Notch signalling pathway is required to establish and maintain the organizing activity at the dorsoventral boundary (D/V boundary). At early stages, the activity of the pathway is restricted to a small stripe straddling the D/V boundary, and the establishment of this activity domain requires the secreted molecule Fringe (Fng). The activity domain will be established symmetrically at each side of the boundary between Fng-expressing and non-expressing cells. Evidence is presented that the Drosophila tumor-suppressor gene lethal giant discs (lgd), a gene whose coding region has yet to be identified, is required to restrict the activity of Notch to the D/V boundary. In the absence of lgd function, the activity of Notch expands from its initial domain at the D/V boundary. This expansion requires the presence of at least one of the Notch ligands, which can activate Notch more efficiently in the mutants. The results further suggest that Lgd appears to act as a general repressor of Notch activity, because it also affects vein, eye, and bristle development (Klein, 2003).

As expected, Dl is strongly activated in lgd mutant clones. This observation raises the possibility that lgd is a negative regulator of expression of Dl. Such a function of lgd would explain the ectopic activation of the Notch pathway in lgd mutant imaginal discs and clones. Alternatively, Dl is also a target of the Notch pathway, and hence the strong ectopic expression of Dl in the mutant clones could be a consequence of the activation of the Notch pathway rather than its initial cause. Two experiments argue for the second alternative. Clones double mutant for lgd and Su(H) fail to express Dl, indicating that a functional Notch pathway is required for expression of Dl in lgd mutant cells. Furthermore, Dl expression is strongly reduced in Su(H) mutant clones induced in lgd mutant wing imaginal discs. Both results indicate that the ectopic expression of Dl is not the cause but a consequence of the activation of the Notch pathway in the wing imaginal disc of lgd mutants. In agreement with this conclusion is the fact that Dl is not activated in lgd mutant clones located in the hinge region. This suggests that expression of Dl is not a consequence of loss of lgd function in all regions of the disc (Klein, 2003).

Atrophin contributes to the negative regulation of epidermal growth factor receptor signaling in Drosophila as measured by the high levels of the known EGFR target gene Dl in Atrophin mutants

Dentato-rubral and pallido-luysian atrophy (DRPLA) is a dominant, progressive neurodegenerative disease caused by the expansion of polyglutamine repeats within the human Atrophin-1 protein. Drosophila Atrophin and its human orthologue are thought to function as transcriptional co-repressors. Drosophila Atrophin participates in the negative regulation of Epidermal Growth Factor Receptor (EGFR) signaling both in the wing and the eye imaginal discs. In the wing pouch, Atrophin loss of function clones induces cell autonomous expression of the EGFR target gene Delta, and the formation of extra vein tissue, while overexpression of Atrophin inhibits EGFR-dependent vein formation. In the eye, Atrophin cooperates with other negative regulators of the EGFR signaling to prevent the differentiation of surplus photoreceptor cells and to repress Delta expression. Overexpression of Atrophin in the eye reduces the EGFR-dependent recruitment of cone cells. In both the eye and wing, epistasis tests show that Atrophin acts downstream or in parallel to the MAP kinase rolled to modulate EGFR signaling outputs. Atrophin genetically cooperates with the nuclear repressor Yan to inhibit the EGFR signaling activity. Finally, it was found that expression of pathogenic or normal forms of human Atrophin-1 in the wing promotes wing vein differentiation and these forms act as dominant negative proteins inhibiting endogenous fly Atrophin activity (Charroux, 2006).

Four facts are evidence that Atro contributes to the negative regulation of EGFR signaling: (1) clones mutant for Atro display phenotypes characteristic of overactive EGFR signaling and express high levels of the known EGFR target gene Dl. These effects are enhanced when negative regulators of EGFR signaling, such as Argos, are simultaneously removed in Atro clones. (2) Increased amounts of Atro reduce the activity of EGFR signaling; (3) ectopic expression of Atro enhances the effects of decreased EGFR signaling, whereas reduced Atro enhances the effects of ectopic signaling and (4) Atro genetically interacts with yan suggesting that both repressors may cooperate to block EGFR signaling output (Charroux, 2006).

The likely C. elegans orthologue of Atrophin, Egl27, has been shown to inhibit vulval development induced by the Ras signal transduction pathway. Thus, the role of Atro as a negative regulator of the RTK/EGFR pathway may have been conserved during evolution. Egl27 is a component of a repressor complex, the nucleosome remodeling and histone deacetylase (NURD) complex, which is composed of HDAC-1, HDAC-2, two proteins of the Mi-2/CHD family, and MTA1 or MTA2. During vulval induction, the NURD complex is proposed to interact with the sequence-specific transcription factors LIN-31, an Ets-related transcription factor and LIN-1, a winged-helix molecule. LIN-1 and LIN-31 are repressors of vulval development that are negatively regulated upon phosphorylation by the MAPK mpk1/sur-1 (Charroux, 2006).

MAPK-dependent phosphorylation of the ETS transcription factor Pnt is necessary for the activation of the EGFR target genes in third instar eye imaginal discs and in embryos. Yan and Atro show synergistic genetic interaction, suggesting that both are required for the repression of EGFR signaling function. Thus, by analogy with EGL-27 and LIN-31 from C. elegans, a model is proposed where Yan cooperates with Atro in order to achieve tight repression. How does EGFR signaling counteract Atro-mediated repression? Localized downregulation (such as nuclear export and/or protein degradation) of specific repressors is a common mechanism for the activation of target genes by the EGFR pathway. Two observations argue against this mechanism for the co-repressor Atro: (1) in cells with high levels of EGFR activity, such as either side of the dorso-ventral boundary in the wing pouch, or later in prospective veins of pupal wings, Atro protein is detected ubiquitously and at invariant levels in all nuclei and (2) when EGFR signaling is overactivated in clones (by expressing the constitutive form of EGFR, EGFRACT), the amount and/or subcellular localization of the co-expressed Atro protein is unchanged (Charroux, 2006).

Several lines of evidence show that, in the late phases of imaginal disc patterning, Atro plays a specific role for EGFR repression. It was found that Atro does not contribute to other signaling pathways during imaginal disc development. For instance, expression of both Distal-less and the vestigial quadrant enhancer (vgQE), two known wingless (wg) target genes, is not affected in Atro clones located in the wing pouch. Plus, it was found that signaling from the Notch (N) receptor does not require Atro activity since Atro clones expressing the constitutively active, intra-cellular fragment of the N receptor (Nintra) display identical phenotypes to Nintra control clones, when located in the wing pouch (Charroux, 2006).

Other signaling pathways are known to affect vein differentiation such as Decapentaplegic (DPP), which promotes vein differentiation in late pupae, and N whose activity is necessary to restrict vein territories. However, the idea is favored that Atro contributes mainly to EGFR signaling since Atro acts in third instar larvae and is dispensable for N activity in the wing (Charroux, 2006).

Despite the strong correlation of Atro repression of EGFR target genes in the imaginal discs, Atro is required for patterning where EGFR has not been implicated. For example, Atro is required for normal segmentation of the Drosophila embryo. However, it is noted that both EGFR signaling and Atro are required for cell survival during embryogenesis. Additionally, Atro is not required for all EGFR-dependent events. For example, Atro is not involved in the function of the EGFR defining the identity of the proximal wing disc. These observations indicate that variable mechanisms of control are implicated in the negative regulation of EGFR signaling in the nucleus (Charroux, 2006).

This notion is supported even in different imaginal tissues. EGFR signals via Strawberry notch (Sno) and Ebi, to inhibit the repressor activity of a Su(H)/SMRTER complex, leading to activation of Dl expression. Clones of cells mutant for the Su(H)SF8 hypomorphic allele cause high level expression of Dl in PR cells, but not in the wing pouch. It was found that clones of cells mutant for the Su(H)del47 null allele similarly do not show ectopic expression of Dl in the wing pouch. As expected, Su(H)del47 cells located at the D/V border abolish the expression of Cut. Thus, Su(H), unlike Atro, is dispensable for Dl repression in the wing pouch. The reverse is true in the eye, where Su(H) activity is absolutely required to repress Dl expression whereas Atro is less important. This is in agreement with the weak phenotype caused by the Atro clones in the eye (i.e. no ectopic PRs, few extra cone cells), and indicates a redundancy with other negative regulators of EGFR signaling. This distinction between the relative requirements in different tissues for different regulators of EGFR signaling provides an interesting insight into tissue-specific control of ubiquitous signaling pathways. Regulators such as Atro, with functions restricted to some tissues, may contribute to the diverse outcomes of signaling through these common pathways (Charroux, 2006).

Dentatorubral-pallidoluysian atrophy (DRPLA) is a dominant, hereditary malady typified by the degeneration of specific neurons in the brain. Although DRPLA has been mimicked in a mouse model, the molecular and cellular mechanisms leading to the disease remain obscure. The data point to the role of Atro in the repression of EGFR signaling. It was found that expression of human N917Atrophin-1 in the wing mimics the loss of Atro activity; this raises the possibility that N917Atrophin-1 is acting as a dominant negative. Additionally, this phenotype is independent of polyQ expansion and is sensitive to the dose of EGFR signaling components. Such effects are not seen following expression of polyQ repeats alone or the exon 1 of Huntingtin with expanded polyQ (93Q) in the wing, indicating that human N917Atrophin-1 has specific effects on this pathway. This mechanistic insight into the role of the fly gene may have broader implications concerning Atrophin function in other organisms (Charroux, 2006).

Regulation of Delta in the eye

Planar polarity is seen in epidermally derived structures throughout the animal kingdom. In the Drosophila eye, planar polarity is reflected in the mirror-symmetric arrangement of ommatidia (eye units) across the dorsoventral midline or equator; ommatidia on the dorsal and ventral sides of the equator exhibit opposite chirality. Photoreceptors R3 and R4 are essential in the establishment of the polarity of ommatidia. The R3 cell is thought to receive the polarizing signal, eminating from the equator, through the receptor Frizzled (Fz), before or at higher levels than the R4 cell, generating a difference between neighbouring R3 and R4 cells. Both loss-of-function and overexpression of Fz in the R3/R4 pair result in polarity defects and loss of mirror-image symmetry. Notch and Delta (Dl) are identified as dominant enhancers of the phenotypes produced by overexpression of fz and dishevelled (dsh); dsh encodes a signaling component downstream of Fz, and it is shown that Dl-mediated activation of Notch is required for establishment of ommatidial polarity. Whereas fz signaling is required to specify R3, Notch signaling induces the R4 fate. These data indicate that Dl is a transcriptional target of Fz/Dsh signaling in R3, and Dl activates Notch in the neighboring R4 precursor. This two-tiered mechanism explains how small differences in the level and/or timing of Fz activation reliably generate a binary cell-fate decision, leading to specification of R3 and R4 and ommatidial chirality. How Notch signaling induces the R4 fate remains unclear, as it usually represses photoreceptor development at this stage. However, the precursor cells are already committed to form the R3/R4 pair by transcription factors (such as Seven-up) that are required for both R3/R4-cell fate and polarity generation (Fanto, 1999).

The small GTPases Rac and Rho act as cellular switches in many important biological processes. In the fruit fly Drosophila, RhoA participates in the establishment of planar polarity, a process mediated by the receptor Frizzled (Fz). Thus far, analysis of Rac in this process has not been possible because of the absence of mutant Rac alleles. The roles of Rac and Rho in establishing the polarity of ommatidia in the Drosophila eye were investigated. By expressing a dominant negative or a constitutively activated form of Rac1, Rac signaling was interfered with specifically and ommatidial polarity was disrupted. The resulting defects are similar to the loss/gain-of-function phenotypes typical of tissue-polarity genes. Through genetic interaction and rescue experiments involving a polarity-specific, loss-of-function dishevelled (dsh) allele, Rac1 was found to act downstream of Dsh in the Fz signaling pathway, but upstream of, or in parallel to, RhoA. Rac signals to the nucleus through the Jun N-terminal kinase (JNK) cascade in this process. By generating point mutations in the effector loop of RhoA, it was found that RhoA also signals to the nucleus during the establishment of ommatidial polarity. Nevertheless, Rac and RhoA activate transcription of distinct target genes. Thus Rac is specifically required downstream of Dsh in the Fz pathway. It functions upstream or in parallel to RhoA and both signal to the nucleus, through distinct effectors, to establish planar polarity in the Drosophila eye (Fanto, 2000).

To better characterize nuclear signaling by Rac and RhoA, the expression of puckered (puc) and Delta (Dl) were studied. Dl is the only known transcriptional target of Fz signaling in R3, and puc-lacZ expression serves as a measure of JNK activity in vivo. The puc gene is a transcriptional target of JNK signaling in Drosophila, and encodes a dual specificity protein phosphatase that acts as a negative regulator of JNK itself in a feedback loop. In the wild type, very weak beta-galactosidase expression from the puc enhancer trap line is detectable in all photoreceptor precursors. Expression of sev;racV12 lead to strong upregulation of puc-lacz in one or, more frequently, two cells of the cluster, identified as R3/R4 precursor cells, consistent with the expression pattern of sev;RacV12. These data resemble the upregulation of puc-lacz when the JNK pathway has been activated in the same cells (Fanto, 2000).

In contrast, RhoV14 affects puc-lacz expression differently. Although in sev;RhoV14 eye discs puc-lacz expression is upregulated in some cells at a later stage, these were not identifiable as the R3/R4 pair, but were often found in the position of the R2/R5/R8 precursors (where sev is not expressed). This suggests that the effect seen is not a direct consequence of Rho activation, but more likely a secondary effect (RhoAV14 E40L fails to induce significant puc-lacz expression). Thus, the direct transcriptional activation of puc-lacz in R3/R4 correlates with the genetic interactions with the JNK module, suggesting a difference in the action of Rac and RhoA (Fanto, 2000).

An important aspect of R3/R4 cell fate and ommatidial polarity determination is the upregulation of Dl expression in the R3 precursor by Fz. Dl then signals to Notch on the R4 precursor, resulting in the choice of the R4 cell fate. In addition to Fz, other components of the Fz/planar-polarity pathway have also been found to upregulate Dl transcription. Thus, whether Rac and RhoA also regulate Dl transcription was investigated by monitoring Dl-lacZ expression in sev;RacV12 and sev;RhoV14 eye discs (Fanto, 2000).

In the wild type, Dl is expressed dynamically in photoreceptor precursors behind the furrow. Within the R3/R4 pair, it is expressed in R3 from rows 4 to 8, whereas it remains at lower levels in R4. In contrast to the difference in puc expression, both sev;RacV12 and sev;RhoV14 upregulated Dl-lacz expression in both R3/R4 precursors. The RhoAV14 E40L isoform that is impaired in nuclear signaling does not affect Dl expression, confirming the importance of nuclear signaling by RhoA. These effects are very similar to those of sev;Fz, supporting the idea that Rac and RhoA act downstream of Fz in the regulation of the R3/R4 cell fate. Their different effects on puc-lacz indicate that their downstream effectors in nuclear signaling are distinct (Fanto, 2000).

Jun acts as a signal-regulated transcription factor in many cellular decisions, ranging from stress response to proliferation control and cell fate induction. Genetic interaction studies have suggested that Jun and JNK signaling are involved in Frizzled (Fz)-mediated planar polarity generation in the Drosophila eye. However, simple loss-of-function analysis of JNK signaling components does not show comparable planar polarity defects. To address the role of Jun and JNK in Fz signaling, a combination of loss- and gain-of-function studies has been used. Like Fz, Jun affects the bias between the R3/R4 photoreceptor pair that is critical for ommatidial polarity establishment. Detailed analysis of jun- clones reveals defects in R3 induction and planar polarity determination, whereas gain of Jun function induces the R3 fate and associated polarity phenotypes. Affecting the levels of JNK signaling by either reduction or overexpression leads to planar polarity defects. Similarly, hypomorphic allelic combinations and overexpression of the negative JNK regulator Puckered causes planar polarity eye phenotypes, establishing that JNK acts in planar polarity signaling. The observation that Delta transcription in the early R3/R4 precursor cells is deregulated by Jun or Hep/JNKK activation, reminiscent of the effects seen with Fz overexpression, suggests that Jun is one of the transcription factors that mediates the effects of fz in planar polarity generation (Weber, 2000).

The Drosophila eye disc is a sac of single layer epithelium with two opposing sides: the peripodial membrane (PM) and the disc proper (DP). Retinal morphogenesis is organized by Notch signaling at the dorsoventral (DV) boundary in the DP. Functions of the PM in coordinating growth and patterning of the DP are unknown. The secreted proteins Hedgehog, Wingless, and Decapentaplegic are expressed in the PM. From there they control DP expression of the Notch ligands Delta and Serrate. Peripodial clones expressing Hedgehog induce Serrate in the DP while loss of peripodial Hedgehog disrupts disc growth. Furthermore, PM cells extend cellular processes to the DP. Therefore, peripodial signaling is critical for eye pattern formation and may be mediated by peripodial processes (Cho, 2000).

Restricted localization of Hh-, Wg-, and Dpp-LacZ+ expressing cells along the DV axis in L1 discs suggests that these signals might act upstream of N. To test this idea, Hh activity was removed using a temperature-sensitive allele; Wg was ectopically expressed using hs-wg, or Dpp activity was removed by using a heteroallelic combination of the two dpp alleles, and then the expression patterns of the N ligands Dl and Ser were visualized in the eye discs. In L2 wild-type discs, Dl is preferentially expressed in the dorsal domain, while Ser is enriched along the DV midline of the DP. Both Dl and Ser are also present in the PM at a low level. In hhts2 mutants shifted to restrictive temperature during the early L1 stage, both Dl and Ser are uniformly expressed in dorsal and ventral domains. Ubiquitous Wg overexpression causes variable defects in Dl pattern such as significant reduction in the dorsal domain except near the margin or mislocalization to the ventral domain. Wg overexpression also causes mislocalization of Ser to the dorsal DP. dppe12/dppd14 mutant discs showed similar disruption of the DV-specific Dl and Ser pattern, indicating the necessity of Hh, Wg, and Dpp in DV patterning (Cho, 2000).

The complex interplay of Hh, Wg, and Dpp signaling has been studied for initiation and progression of the morphogenetic furrow. This study has examined much earlier stages of eye development to determine whether these same molecules organize DV patterning prior to retinal differentiation; it has been demonstrated that: (1) Hh, Wg, and Dpp display distinct DV expression patterns in the PM in early discs; (2) their signals are essential for domain-specific expression of Dl and Ser in the DP, and (3) signaling from the PM to DP is important for patterning in the DP. These findings provide a novel view of how eye discs are patterned, a model suggesting Hh, Wg, and Dpp signal from the PM to the DP by means of cellular processes (Cho, 2000).

Recent studies have shown that disc cells send out long and thin cytoplasmic extensions, named cytonemes. Cytonemes are actin-based extensions that grow from the apical surface of the DP cells toward the signaling center, the anterior-posterior boundary of the wing disc. Some of the peripodial extensions described in this study also show cytoneme-like long and thin processes, although it is not known whether the processes are also actin-based. The peripodial processes observed can be readily seen in fixed tissues, unlike cytonemes that cannot be detected in fixed discs. Furthermore, cytonemes extend from the DP cells and grow on the apical surface of the DP, while peripodial processes extend from the apical surface of PM cells across the disc lumen to the DP. In addition, the observation of different shapes of processes suggests that peripodial processes exist in multiple types (Cho, 2000).

Inductive signaling between two cell layers is an important mechanism of morphogenesis in vertebrate development. For instance, BMP4 signaling between optic vesicle and surface ectoderm is important for lens induction in vertebrates. Wnt signaling between the ectoderm and the mesoderm is also crucial for proper dorsoventral limb patterning. First shown to occur during Drosophila leg disc regeneration and now in the eye, peripodial signaling to the DP may be analogous to such inductive signaling in vertebrates. This study illustrates a novel mechanism of interepithelial signaling between PM and DP layers and its importance in eye disc patterning. Significantly, ablation or genetic disruption of the PM also affects development of the DP, providing additional evidence for peripodial signaling. Precise localization of receptors and downstream components for Hh, Wg, and Dpp in early eye discs will help in understanding how these signals are transmitted between the PM and the DP (Cho, 2000).

The hindsight gene regulates cell morphology, cell fate specification, planar cell polarity and epithelial integrity during Drosophila retinal development. In the third instar larval eye imaginal disc, Hnt protein expression begins in the morphogenetic furrow and is refined to cells in the developing photoreceptor cell clusters just before their determination as neurons. In hnt mutant larval eye tissue, furrow markers persist abnormally, posterior to the furrow; there is a delay in specification of preclusters as cells exit the furrow; there are morphological defects in the preclusters, and recruitment of cells into specific R cell fates often does not occur. Additionally, genetically mosaic ommatidia with one or more hnt mutant outer photoreceptor cell, have planar polarity defects that include achirality, reversed chirality and misrotation. Mutants in the JNK pathway act as dominant suppressors of the hnt planar polarity phenotype, suggesting that Hnt functions to downregulate JUN kinase (JNK) signaling during the establishment of ommatidial planar polarity. Hnt expression continues in the photoreceptor cells of the pupal retina. When an ommatidium contains four or more hnt mutant photoreceptor cells, both genetically mutant and genetically wild-type photoreceptor cells fall out of the retinal epithelium, indicating a role for Hnt in maintenance of epithelial integrity. In the late pupal stages, Hnt regulates the morphogenesis of rhabdomeres within individual photoreceptor cells and the separation of the rhabdomeres of adjacent photoreceptor cells. Apical F-actin is depleted in hnt mutant photoreceptor cells before the observed defects in cellular morphogenesis and epithelial integrity. The analyses presented here, together with previous studies in the embryonic amnioserosa and tracheal system, show that during development Hnt has a general role in regulation of the F-actin-based cytoskeleton, JNK signaling, cell morphology and epithelial integrity (Pickup, 2002).

The fact that all of the symmetrical ommatidia along the borders of hnt clones are of the R3/R3 conformation suggests that Hnt function is necessary for correct R4 fate and orientation. It has been suggested that, owing to its closer proximity to the polarizing signal from the equator, a stronger activation of the JNK pathway occurs in the R3 precursor cell. Activated JUN would then be responsible for the upregulation of the target gene, Delta, in the R3 precursor cell relative to the R4 precursor cell. Since results in the eye and results in the embryo imply that Hnt is necessary for downregulating JNK function, it is proposed that the wild-type function of Hnt is to downregulate JNK activity in the R4 precursor cell. Such downregulation would enhance JNK signaling differences between the R3 and R4 cells. In the absence of the Hnt gene product, JNK signaling would be inappropriately elevated in the R4 precursor cell, thereby upregulating the transcription of JNK targets such as Delta, leading that cell to behave more like an R3 precursor cell. Consistent with this model, it has been found that Delta hypomorphs act as enhancers of the hntpeb rough eye phenotype. R3/R3 symmetric clusters are observed both when the R4 cell is mutant for hnt and the R3 precursor is hnt+, and when the R3 cell is mutant for hnt and the R4 precursor is Hnt+. In the latter case, the above model would lead one to expect normal R3/R4 clusters. Since only R3/R3 clusters are observed, it is speculated that Hnt can affect the R4 precursor cell when expressed only in the neighboring R3 precursor cell (i.e. that there may be some communication feedback between these cells leading to local non-autonomy of the hnt phenotype) (Pickup, 2002).

Planar cell polarity is established in the Drosophila eye through distinct fate specification of photoreceptors R3 and R4 by a two-tiered mechanism employing Fz and Notch signaling: Fz signaling specifies R3 and induces Dl to activate Notch in R4. The atypical cadherin Flamingo (Fmi) plays critical, but distinct, roles in both R3 and R4. Fmi is first enriched at equatorial cell borders of R3/R4, positively interacting with Fz/Dsh. Subsequently, Fmi is upregulated in R4 by Notch and functions to downregulate Dl expression by antagonizing Fz signaling. This in turn amplifies and enforces the initial Fz-signaling bias in the R3/R4 pair. These results reveal differences in the planar cell polarity genetic circuitry between the eye and the wing (Das, 2002).

To investigate the role of flamingo in eye development, fmi mutant clones were induced with the eye specific ey-FLP/FRT system. Analysis of fmi- tissue in adult eyes shows typical PCP defects with randomized chirality, resulting in loss of mirror image symmetry. Reminiscent of fz, dsh, and stbm null alleles, fmi- clones display defects in ommatidial chirality establishment (random chirality and symmetrical clusters) and rotation. In addition, fmi- clones contain ~20% ommatidia lacking photoreceptors (Das, 2002).

PCP aspects of the fmi phenotype are apparent from the earliest stage in the five-cell precluster. All markers reflecting the arrangement and rotation of ommatidial preclusters (Spalt: R3/R4; Bar: R1/6; svp-lacZ: R3/R4 and R1/R6) show typical polarity defects in fmi- tissue, with a random selection of the direction of rotation and abnormal rotation degrees. Thus, PCP defects are the primary phenotypic features of fmi clones, confirming its critical role in ommatidial polarity establishment (Das, 2002).

During eye disc patterning, Fmi protein is localized apically in all cells anterior to the morphogenetic furrow (MF), within the MF, and in a few rows of developing ommatidia posterior to the MF. Subsequently, Fmi is detected in differentiating photoreceptor cells in perinuclear areas and growing axons, possibly reflecting a late function of fmi in photoreceptor differentiation (Das, 2002).

The initial asymmetrical enrichment of Fmi in both R3 and R4, and the subsequent enrichment in R4 only, raised the question of in which cell(s) of the precluster is fmi required for PCP establishment. Interestingly, the analysis of mosaic clusters revealed a requirement for fmi in both R3 and R4. An ommatidium always adopts the correct orientation when both R3 and R4 are fmi+. When either R3 or R4 (or both) are fmi-, the ommatidium selects chirality randomly or stays symmetrical. Significantly, all ommatidia with wrong or no chirality had fmi- R3 and/or R4 cells. Loss of fmi function in any other R cells in any combination has no effect on ommatidial polarity. These data indicate that fmi is necessary and sufficient in both the R3 and R4 photoreceptor precursors for normal polarity establishment (Das, 2002).

The genetic requirement of fmi in both R3 and R4 is unique, since other PCP genes are required only in either cell (fz and dsh in R3 and stbm and N in R4), and raised the question of how Fmi relates to these genes in function and expression. Thus, the expression patterns of other PCP proteins were examined in the eye (Das, 2002).

Although the fmi phenotype is reminiscent of fz and dsh, the role of fmi is more complex: fmi is first required in an Fz-dependent manner in the R3/R4 pair and later is specifically upregulated in R4 (Das, 2002).

An indication for a late role of fmi in R4 and a link to Fz-Notch signaling comes from the sev --> Fmi experiment (resulting in the overexpression of Fmi in the R3/R4 pair) and its endogenous late expression in R4 (Das, 2002).

The sev-Fmi mosaics give insight into the Fmi function at this stage. In mosaic sev-Fmi R3/R4 pairs, the cell with higher Fmi levels adopts the R4 fate. However, when both cells have equally high levels of Fmi, the cluster has a high tendency to develop as an R3/R3 symmetrical ommatidium. How could this be explained? The sev --> Fmi genotype is both sensitive to Dl dosage and causes a downregulation of Dl transcription in R3/R4. As Dl is nonautonomously required for R4 induction, it serves as a link between the mosaic requirement and phenotypic features of sev --> Fmi: when both cells contain sev --> Fmi, Dl is downregulated in both cells, and Notch activation often fails in both cells of the pair. This leads to a lack of R4 induction, generating R3/R3 symmetrical ommatidia. However, when only one cell of the pair has high Fmi levels, Dl is kept lower in this cell (even if it was originally the R3 precursor). It then adopts an R4 fate because it receives higher Dl levels from its neighbor. Thus, the endogenous role of Fmi in R4 could be to inhibit Dl expression and enhance the differences in Dl levels between R3 and R4 (Das, 2002).

How does Fmi inhibit Dl expression in R4? This is likely mediated through Fz signaling, as it was speculated in PCP establishment in the wing that Fmi can antagonize Fz. Thus, Fmi-mediated inhibition of Fz/PCP signaling should be a general mechanism. The lack of genetic interactions between fz and fmi (in either direction) suggests that Fmi does not directly inhibit Fz, and the mechanistic dissection of this inhibition will require biochemical studies (Das, 2002).

Notch activation and Fmi upregulation are coincident in R4, and, thus, Notch signaling itself is a good candidate for Fmi upregulation. The sev-N* (expression of activated Notch using a sevenless promoter) data indicate that Notch activation indeed leads to Fmi upregulation in a cell autonomous manner: all cells that express the sev enhancer, including the nonneuronal cone cells normally not expressing Fmi significantly, show an upregulation. Since nuclear Notch (N-intra) shows the same effect, it is likely a direct transcriptional event (Das, 2002).

These data indicate that the initial two-tiered mechanism of Fz-Notch activation establishing the R3/R4 fates and polarity in the eye can be extended further to include Notch-mediated upregulation of Fmi in R4. This in turn inhibits Fz/PCP-mediated Dl induction in R4 and amplifies the signaling differences between R3 and R4, leading to a solid binary cell fate decision (Das, 2002).

Since Fmi is initially expressed in both cells of the pair, its inhibitory role on Dl can only be allowed in R4 and thus needs to be regulated. How is this achieved? Diego (Dgo) is a good candidate for this role. The cytoplasmic Dgo protein depends on Fmi for membrane association and generally colocalizes with Fmi at all membranes in the eye disc. The genetic interactions with sev --> Fmi identify dgo as a strong enhancer, suggesting that it is suppressing Fmi function in this context. Mosaic analysis of dgo shows that it is required in R3 and thus might keep the inhibitory function of Fmi off in R3. Since Fmi is necessary, but not sufficient, for Dgo membrane recruitment, other factor(s) are also required. Since Dgo and Fmi colocalize also in R4, a factor is needed there to antagonize Dgo function. Strabismus (Stbm), since it is required in R4, is a candidate. Since fmi mutants are enhancers of an Stbm overexpression phenotype, Stbm could serve this function in R4 (Das, 2002).

The Notch and Epidermal growth factor receptor (Egfr) pathways both regulate proliferation and differentiation, and the cellular response to each is often influenced by the other. A mechanism is described that links them in a sequential fashion, in the developing compound eye of Drosophila. Egfr activation induces photoreceptor (R cell) differentiation and promotes R cell expression of Delta. This Notch ligand then induces neighboring cells to become nonneuronal cone cells. ebi and strawberry notch (sno) regulate Egfr-dependent Delta transcription by antagonizing a repressor function of Suppressor of Hairless [Su(H)]. Sno binds to Su(H), and Ebi, an F-box/WD40 protein, forms a complex with Su(H) and the corepressor Smrter. Egfr-activated transcriptional derepression requires ebi and sno, is proteasome-dependent, and correlates with the translocation of Smrter to the cytoplasm (Tsuda, 2002).

The Notch signaling pathway plays multiple roles in eye development. At the morphogenetic furrow, the proneural protein Atonal facilitates the expression of Dl in the R8 cell. The first step of ommatidial assembly involves lateral inhibition between equivalent cells, but successive steps are inductive, arising from an already differentiated cell to its uncommitted neighbors. The Notch pathway is involved in the regulation of both of these processes. Similarly, the Egfr ligand, Spi, expressed in R8, activates the receptor in neighbors allowing them to assume their respective R1–R7 cell fates. Subsequently, these R cells express Spi, and as described in this study, they also express Dl in response to Egfr activation. The cone cells receive an Egfr signal and a Notch signal from the R cells and this combination is critical for the assumption of their fate. Later, after their fate is determined, these cone cells, too, will express Delta, which is important for pigment cell induction. Presumably, the level of the Egfr signal rises in the cone cells with time, and as a threshold of Egfr activation is surpassed, the proteasome mediated arm of the pathway becomes effective causing derepression of Su(H) and expression of functional levels of Dl sufficient for pigment cell development. Thus, a temporally and spatially positioned combination of parallel and sequential Egfr/Notch signals is important for the successive induction of cell types in the eye (Tsuda, 2002).

Evidence from mammalian systems has suggested that CBF1, the mammalian homolog of Su(H), is a component of a large repressor complex. The activation function of CBF1 results from a displacement of repressive components (such as HDAC) by the intracellular domain of Notch which converts Su(H) into a transcriptional activator. Genetic analysis of the embryonic midline and the pupal bristle complexes in Drosophila have also supported a switch from Su(H)-mediated repression to activation. A second mechanism for relieving Su(H) mediated repression is through Sno, Ebi, and the Egfr pathway. In response to the Egfr signal, Ebi, an F-box protein, presumably causes a proteasome-mediated degradation of an unknown component of the Su(H) inhibitory complex. Mammalian TBL1 (Ebi) can function downstream of the tumor suppressor gene, p53, in the degradation of the ß-catenin protein in a novel ubiquitin-dependent degradation pathway involving Siah, the mammalian homolog of the Drosophila Sina protein. Similarly, Drosophila Ebi can also act in combination with Sina to degrade protein targets. More generally, phosphorylation by MAPK downstream of RTK pathways is known to trigger proteasome-mediated degradation of target proteins. In addition to Ebi, a core component of the proteasome, encoded by l(3)73Ai gene, is also important for expression of Dl. The simplest model is that in response to Egfr signaling, one or more of the many components in the large Su(H)/SMRTER repression complex becomes a target of a proteasome-mediated degradation process (Tsuda, 2002).

The studies presented here also show that the corepressor SMRTER is redistributed from the nucleus to the cytoplasm in an Egfr/Sno/Ebi dependent manner. These results are in complete agreement with the role of the corresponding mammalian protein SMRT in its function as a repressor. Like Su(H), nuclear hormone receptors such as retinoic acid receptor and thyroid hormone receptor can function as both repressors and activators. SMRT has been shown to be phosphorylated in response to an RTK signal. This leads to translocation of SMRT out of the nucleus. Thus, steroid hormone receptors lose their ability to repress but not activate transcription. In an in vivo example, the Egfr/Sno/Ebi pathway promotes the dissociation of the Su(H)/SMRTER repressor complex and causes the nuclear export of SMRTER. As a result, target genes such as Dl are derepressed (Tsuda, 2002).

Notch signaling can take place between cells that are equivalent at the time the signal initiates, or it can occur between a signaling cell that is different from the cell receiving the signal. Traditionally, the first kind of process has been referred to as lateral inhibitory Notch signaling and the second as an inductive Notch pathway. These studies suggest that the fundamental difference between these two processes is not due to differences in molecular components of the pathway downstream from activated Notch, but rather due to the mechanism that controls the expression of the ligand, Dl. For lateral inhibitory Notch pathways, a mechanism involving a feedback loop and proneural genes is at the core of Dl/Notch regulation. Starting with an equipotent group, an asymmetric signaling system is created, in which the signaling cell expressing high levels of Dl, assumes a differentiated fate and prevents its neighbors from adopting an identical fate. All available evidence suggests that the Egfr pathway, Sno, and Ebi do not control Dl expression in such lateral inhibitory processes mediated by Notch. In contrast, this study shows that in inductive processes controlled by Notch signaling, Dl expression is controlled by Egfr, Ebi, and Sno and apparently not by proneural genes. For example, no known proneural gene (Ac/sc, amos, or atonal) is expressed in R cells that contact the cone cells (i.e., R1-R7) and express Dl. This is also true for cells at the dorsoventral boundary of the wing disc where Notch signaling directly activates vestigial expression through Su(H) binding to the enhancer and in the mesectodermal cells of stage 6 embryos where the Notch pathway has been implicated in controlling the expression of single minded at the midline. Instead, all of these cells in the eye, wing, and embryo receive an Egfr signal that likely controls Dl expression. Indeed, the late expression of Dl in R cells does not involve feedback from the cone cells but instead involves the derepression of Dl expression in a Notch-independent manner. This is different from the early expression of Dl that is required for the selection of R8 cells at the furrow through a lateral inhibitory signal (Tsuda, 2002).

This study highlights the function of two unusual proteins, Sno and Ebi, in controlling the expression of Dl. Mammalian Ebi (TBL1) interacts with a SMRT/HDAC complex as also supported by this study in Drosophila. There are two human and three mouse genes similar to Sno identified by genome projects. The function of the mammalian Sno proteins is unknown. Whether the mammalian proteins also function upstream of the Notch pathway, as they do in Drosophila, remains to be established. Given the conservation of developmental pathways between Drosophila and mammals, this may not be an unreasonable expectation (Tsuda, 2002).

The establishment of planar cell polarity in the Drosophila eye requires correct specification of the R3/R4 pair of photoreceptor cells. In response to a polarizing factor, Frizzled signaling specifies R3 and induces Delta, which activates Notch in the neighboring cell, specifying it as R4. The spalt zinc-finger transcription factors (spalt major and spalt-related) are part of the molecular mechanisms regulating R3/R4 specification and planar cell polarity establishment. In mosaic analysis, spalt genes have been shown to be specifically required in R3 for the establishment of correct ommatidial polarity. In addition, spalt genes are required for proper localization of Flamingo in the equatorial side of R3 and R4, and for the upregulation of Delta in R3. These requirements are very similar to those of frizzled during R3/R4 specification. spalt genes are required cell-autonomously for the expression of seven-up in R3 and R4, and seven-up is downstream of spalt genes in the genetic hierarchy of R3/R4 specification. Thus, spalt and seven-up are necessary for the correct interpretation of the Frizzled-mediated polarity signal in R3. Finally, it has been shown that, posterior to row seven, seven-up represses spalt in R3/R4 in order to maintain the R3/R4 identity and to inhibit the transformation of these cells to the R7 cell fate (Domingos, 2004).

In conclusion, these results demonstrate that sal is required in R3 to allow normal Fz/PCP signaling to specify the R3 and R4 cell fates. Ommatidia mutant for sal show defects that are very similar to those observed in fz and dsh mutants, as judged by the loss of asymmetric Fmi localization at the equatorial side of the R3/R4 precursors, and by the lack of Dl and E(spl)mdelta upregulation within the R3/R4 pair. In addition, sal is required upstream of svp for normal R3/R4 specification. Finally, these results show that, posterior to row seven, svp represses sal in R3/R4 in order to maintain R3/R4 identity and to inhibit transformation of these cells to the R7 cell fate (Domingos, 2004).

In the developing eye of Drosophila, the EGFR and Notch pathways integrate in a sequential, followed by a combinatorial, manner in the specification of cone-cell fate. This study demonstrates that the specification of primary pigment cells requires the reiterative use of the sequential integration between the EGFR and Notch pathways to regulate the spatiotemporal expression of Delta in pupal cone cells. The Notch signal from the cone cells then functions in the direct specification of primary pigment-cell fate. EGFR requirement in this process occurs indirectly through the regulation of Delta expression. Combined with previous work, these data show that unique combinations of only two pathways -- Notch and EGFR -- can specify at least five different cell types within the Drosophila eye (Nagaraj, 2007).

Unlike photoreceptor R cells, cone cells do not express Delta at the third instar stage of development. However, these same cone cells express Delta at the pupal stage. In addition, correlated with this Delta expression, the upregulation of phosphorylated MAPK was observed in these cells. This is very similar to the earlier events seen in R cells during larval development, in which the activation of MAPK causes the expression of Delta. Also, as in the larval R cells, the pupal upregulation of Delta in cone cells is transcriptional. A Delta-lacZ reporter construct, off in the larval cone cell, is detected in the corresponding pupal cone cells. To determine whether EGFR is required for the activation of Delta in the pupal cone cells, the temperature-sensitive allele EGFRts1 was used. Marked clones of this allele were generated in the eye disc using ey-flp at permissive conditions and later, in the mid-pupal stages, shifted the larvae to a non-permissive temperature. Cells mutant for EGFR, but not their adjacent wild-type cells, showed a loss of Delta expression. However, both mutant and wild-type tissues showed normal cone-cell development, as judged by Cut (a cone-cell marker) expression. As supporting evidence, ectopic expression of a dominant-negative version of EGFR (EGFRDN) in cone cells using spa-Gal4 after the cells have already undergone initial fate specification also causes a complete loss of Delta expression without compromising the expression of the cone-cell-fate-specification marker (Nagaraj, 2007).

Gain-of-function studies further support the role of EGFR signaling in the regulation of Delta expression in cone cells. Although weak EGFR activation is required for cone-cell fate, activated MAPK is not detectable in cone-cell precursors of the third instar larval eye disc. When spa-Gal4 (prepared by cloning the 7.1 kb EcoRI genomic fragment of D-Pax2) is used to express an activated version of EGFR in larval cone cells, detectable levels of MAPK activation in these cells were found and the consequent ectopic activation of Delta in the larval cone cells occurred. Taken together, these gain- and loss-of-function studies show that, during pupal stages, EGFR is required for the activation of Delta. However, this Delta expression is not essential for the maintenance of cone-cell fate (Nagaraj, 2007).

In larval R cells, the activation of Delta transcription in response to EGFR signaling is mediated by two novel nuclear proteins, Ebi and Sno. To determine the role of these genes in wild-type pupal-cone-cell Delta expression, sno and ebi function were selectively blocked in the pupal eye disc. A heteroallellic combination of the temperature-sensitive allele snoE1 and the null allele sno93i exposed to a non-permissive temperature for 12 hours caused a significant reduction in Delta expression. Similarly, a dominant-negative version of ebi also caused the loss of Delta expression. Importantly, pupal eye discs of neither spa-Gal4, UAS-ebiDN nor snoE1/sno93i showed any perturbation in cone-cell fate, as judged by the expression of Cut. Thus, as in the case of larval R cells, the loss of ebi and sno in the pupal cone cells causes the loss of Delta expression without causing a change in cone-cell fate (Nagaraj, 2007).

To test whether the expression of Delta in pupal cone cells is required for the specification of primary pigment cells, Nts pupae were incubated at a non-permissive temperature for 10 hours during pupal development and pigment-cell differentiation was monitored using BarH1 (also known as Bar) expression as a marker. Loss of Notch signaling during the mid-pupal stages caused a loss of Bar, further demonstrating the requirement of Notch signaling in the specification of primary pigment-cell fate. Similarly, when the 54CGal4 driver line, which is activated in pigment cells, was used to drive the expression of a dominant-negative version of Notch, pupal eye discs lost primary pigment-cell differentiation, again suggesting an autonomous role for Notch in pigment-cell precursors. In neither the Nts nor the 54C-Gal4, UAS-NDN genetic background was perturbation observed in cone-cell fate specification. It is concluded that Delta activation mediated by EGFR-Sno-Ebi in pupal cone cells is essential for neighboring pigment-cell fate specification (Nagaraj, 2007).

Delta-protein expression in pupal cone cells is initiated at 12 hours and is downregulated by 24 hours of pupal development. To determine the functional significance of this downregulation, the genetic combination spa-Gal4/UAS-Delta was used, in which Delta is expressed in the same cells as in wild type, but is not temporally downregulated. Whereas, in wild type, a single hexagonal array of pigment cells surrounded the ommatidium, in the pupal eye disc of spa-Gal4, UAS-Delta flies, multiple rows of pigment cells were observed surrounding each cluster. Furthermore, in wild type, only two primary pigment cells were positive for Bar expression in each cluster, whereas, in spa-Gal4, UAS-Delta pupal eye discs, ectopic expression of Bar was evident in the interommatidial cells. Therefore, the temporal regulation of Notch signaling and its activation, as well as its precise downregulation, are essential for the proper specification of primary pigment-cell fate (Nagaraj, 2007).

By contrast to the autonomous requirement for Notch signaling in primary pigment cells, the function of the EGFR signal appears to be required only indirectly in the establishment of primary pigment-cell fate through the regulation of Delta expression in the pupal cone cells. When a dominant-negative version of EGFR was expressed using hsp70-Gal4 at 10-20 hours after pupation, no perturbation was observed in the specification of primary pigment cells, as monitored by the expression of the homeodomain protein Bar. By contrast, the expression of dominant-negative Notch under the same condition resulted in the loss of Bar-expressing cells. Thus, in contrast to Notch, blocking EGFR function at the time of primary pigment-cell specification does not block the differentiation of these cells. Importantly, blocking EGFR function in earlier pupal stages caused the loss of Delta expression in cone cells and the consequent loss of pigment cells. Based on these observations, it is concluded that, in the specification of primary pigment-cell fate, the Notch signal is required directly in primary pigment cells, whereas EGFR function is required only indirectly (through the regulation of Delta) in cone cells (Nagaraj, 2007).

The Runt-domain protein Lz functions in the fate specification of all cells in the developing eye disc arising from the second wave of morphogenesis. At a permissive temperature (25°C), lzTS114 pupal eye discs showed normal differentiation of primary pigment cells. lzTS114 is a sensitized background in which the Lz protein is functional at a threshold level. When combined with a single-copy loss of Delta, a dosage sensitive interaction caused the loss of primary pigment cells. By contrast, under identical conditions, a single-copy loss of EGFR function had no effect on the proper specification of primary pigment-cell fate. This once again supports the notion that the specification of primary pigment cells directly requires Lz and Notch, whereas EGFR is required only indirectly to activate Delta expression in cone cells (Nagaraj, 2007).

This study highlights two temporally distinct aspects of EGFR function in cone cells. First, this pathway is required for the specification of cone-cell fate at the larval stage, and EGFR is then required later in the pupal cone cell for the transcriptional activation of Delta, converting the cone cell into a Notch-signaling cell. Delta that was expressed in the cone cell through the activation of the Notch pathway functioned in combination with Lz in a cell autonomous fashion and promoted the specification of the primary pigment-cell fate (Nagaraj, 2007).

Studies using overexpressed secreted Spitz have shown that ectopic activation of the EGFR signal in all cells of the pupal eye disc results in excess primary pigment cells. This study shows that EGFR activation in the pupal eye disc is required for the transcriptional activation of Delta in cone cells, but that the loss of EGFR function at the time when primary pigment cells are specified does not perturb their differentiation. It is concluded that the ectopic primary pigment cells seen in an activated-EGFR background result from the ectopic activation of Delta, which then signals adjacent cells and promotes their differentiation into primary pigment cells. Indeed, it has been shown that excessive Delta activity results in the over specification of primary pigment cells. The results are also consistent with the previous observation that the EGFR target gene Argos is not expressed in primary pigment cells in pupal eye discs. Additionally, loss of EGFR function in pupal eye discs does not perturb the normal patterning of interommatidial bristle development, which develop even later than the primary pigment cells (Nagaraj, 2007).

The elucidation of the Sevenless pathway for the specification of R7 led to the suggestion that different cell types within the developing eye in Drosophila will require combinations of dedicated signaling pathways for their specification. However, studies from several laboratories have suggested that the Sevenless pathway seems to be an exception, in that cell-fate-specification events usually require reiterative combinations of a very small number of non-specific signals. Cone-cell fate is determined by the sequential integration of the EGFR and Notch pathways in R cells followed by the parallel integration of the EGFR and Notch pathways in cone-cell precursors. This study shows that the most important function of EGFR in the specification of primary pigment cells is to promote the transcriptional activation of Delta in cone cells through the EGFR-Ebi-Sno-dependent pathway. The sequential integration of the EGFR and Notch pathways, first used in the larval stage for Delta activation in R cells, is then reused a second time in cone cells to regulate the spatiotemporal expression of Delta, converting the cone cells at this late developmental stage to Notch-signaling cells. Delta present in the cone cell then signals the adjacent undifferentiated cells for the specification of primary pigment cells. For this process, the Notch pathway functions directly with Lz but indirectly with EGFR. Through extensive studies of this system it now seems conclusive that different spatial and temporal combinations of Notch and EGFR applied at different levels can generate all the signaling combinations needed to specify the neuronal (R1, R6, R7) and nonneuronal (cone, pigment) cells in the second wave of morphogenesis in the developing eye disc (Nagaraj, 2007).

The EGFR and Notch pathways are sequentially integrated, in a manner similar to that described here, in multiple locations during Drosophila development. In the development of wing veins, EGFR that is activated in the pro-vein cells causes the expression of Delta, which then promotes the specification of inter-vein cells. Similarly, these two pathways are sequentially integrated in the patterning of embryonic and larval PNS, and during muscle development. Indeed, there are striking similarities between the manner in which the EGFR and Notch pathways are integrated in the developmental program in the C. elegans vulva and the Drosophila eye. During vulval fate specification in the C. elegans hermaphrodite gonad, anchor cells are a source of EGFR signal (Lin3), which induces the specification of the nearest (P6) cell to the primary cell fate from within a group of six equipotent vulval precursor cells (VPC). This high level of EGFR activation induces the transcriptional activation of Notch ligands in the primary cells in what can be considered sequential integration of the two pathways - the Notch signal from the primary cell both inhibits EGFR activity in the VPCS on either side of P6.p and also promotes the secondary cell fate. Thus, the reiterative integration of these two signals, in series and in parallel, can be used successfully to specify multiple cell fates in different animal species. Given that the RTK and Notch pathways function together in many vertebrate developmental systems, it is likely that similar networks will be used to generate diverse cell fates using only a small repertoire of signaling pathways (Nagaraj, 2007).

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).

Role of JAK/STAT signaling in neuroepithelial stem cell maintenance and proliferation in the Drosophila optic lobe

During Drosophila optic lobe development, proliferation and differentiation must be tightly modulated to reach its normal size for proper functioning. The JAK/STAT pathway plays pleiotropic roles in Drosophila development and in the larval brain, has been shown to inhibit medulla neuroblast formation. This finds that JAK/STAT activity is required for the maintenance and proliferation of the neuroepithelial stem cells in the optic lobe. In loss-of-function JAK/STAT mutant brains, the neuroepithelial cells lose epithelial cell characters and differentiate prematurely while ectopic activation of this pathway is sufficient to induce neuroepithelial overgrowth in the optic lobe. It was further shown that Notch signaling acts downstream of JAK/STAT to control the maintenance and growth of the optic lobe neuroepithelium. Thus, in addition to its role in suppression of neuroblast formation, the JAK/STAT pathway is necessary and sufficient for optic lobe neuroepithelial growth (Wang, 2011).

This study has shown that JAK/STAT signaling plays an important role in the maintenance and expansion of the neuroepithelial stem cells in the Drosophila larval optic lobe. Loss of JAK/STAT function leads to the disruption of the adherens junction and disintegration of the OPC neuroepithelium while ectopic JAK/STAT activation is sufficient to generate ectopic neuroepithelial stem cells. The non-cell autonomous induction of NEs in the medulla by ectopic expression of upd strongly suggests that JAK/STAT plays a direct role in the growth and proliferation of neuroepithelial stem cells, rather than promotes growth indirectly by blocking premature differentiation of the NEs into medulla neuroblasts (Wang, 2011).

The Drosophila JAK/STAT pathway has been shown to be essential for stem cell maintenance in the testis and ovary, and in the intestine. This study presents evidence that this pathway is also required for the maintenance and expansion of another type of stem cell, the optic lobe neuroepithelial stem cells. Interestingly, STAT3 activity has been implicated in the maintenance of the neuroepithelial stem cells in the developing mouse brain. Thus, the JAK/STAT pathway may play a conserved role in the maintenance and proliferation of stem cells in general and neural stem cells in particular (Wang, 2011).

The phenotypes of JAK/STAT pathway mutants in the optic lobe are reminiscent of those of Notch mutants. The similar phenotypes of the two pathways indicate that they might interact or cooperate to control optic lobe development. Several recent studies reported the interactions between these two pathways in the optic lobe. It has been proposed that the JAK/STAT pathway acts upstream of Notch because ectopic expression of hopTum-l in clones caused a delay of Dl upregulation on the medial region of the OPC, which suggests that JAK/STAT represses Dl expression. However, this study observed ectopic induction of Dl expression in hopTum-l clones. Others have instead suggested that the two pathways are interdependent and cooperate with one another during optic lobe development, based on the observation that Dl expression was reduced in stat92Ets brains and JAK signaling activity was compromised in Notch signaling mutants that expressed a dominant-negative Su(H) construct, although, late-third instar larval brains were analyzed which should have lost the neuroepithelial cells due to loss of either Notch or JAK signaling activity thus complicating the interpretation of the results. The current data indicate that JAK/STAT signaling stimulates the Notch pathway which controls the maintenance and expansion of the OPC neuroepithelium (Wang, 2011).

Dose-sensitive autosomal modifiers identify candidate genes for tissue autonomous and tissue nonautonomous regulation by the Drosophila nuclear zinc-finger protein, Hindsight

The nuclear zinc-finger protein encoded by the hindsight (hnt) locus regulates several cellular processes in Drosophila epithelia, including the Jun N-terminal kinase (JNK) signaling pathway and actin polymerization. Defects in these molecular pathways may underlie the abnormal cellular interactions, loss of epithelial integrity, and apoptosis that occurs in hnt mutants, in turn causing failure of morphogenetic processes such as germ band retraction and dorsal closure in the embryo. To define the genetic pathways regulated by hnt, 124 deficiencies on the second and third chromosomes and 14 duplications on the second chromosome were assayed for dose-sensitive modification of a temperature-sensitive rough eye phenotype caused by the viable allele, hntpeb; 29 interacting regions were identified. Subsequently, 438 P-element-induced lethal mutations mapping to these regions and 12 candidate genes were tested for genetic interaction, leading to identification of 63 dominant modifier loci. A subset of the identified mutants also dominantly modify hnt308-induced embryonic lethality and thus represent general rather than tissue-specific interactors. General interactors include loci encoding transcription factors, actin-binding proteins, signal transduction proteins, and components of the extracellular matrix. Expression of several interactors was assessed in hnt mutant tissue. Five genes -- apontic (apt), Delta (Dl), decapentaplegic (dpp), karst (kst), and puckered (puc) -- regulate tissue autonomously and, thus, may be direct transcriptional targets of Hnt. Three of these genes -- apt, Dl, and dpp -- are also regulated nonautonomously in adjacent non-Hnt-expressing tissues. The expression of several additional interactors -- viking (vkg), Cg25, and laminin-alpha (LanA) -- is affected only in a nonautonomous manner (Wilk, 2004).

An NRSF/REST-like repressor downstream of Ebi/SMRTER/Su(H) regulates eye development in Drosophila; charlatan is repressed by Ebi/SMRTER and promotes Dl expression

The corepressor complex that includes Ebi and SMRTER is a target of epidermal growth factor (EGF) and Notch signaling pathways and regulates Delta (Dl)-mediated induction of support cells adjacent to photoreceptor neurons of the Drosophila eye. A mechanism is described by which the Ebi/SMRTER corepressor complex maintains Dl expression. charlatan (chn) is repressed by Ebi/SMRTER corepressor complex by competing with the activation complex that includes the Notch intracellular domain (NICD). Chn represses Dl expression and is critical for the initiation of eye development. Thus, under EGF signaling, double negative regulation mediated by the Ebi/SMRTER corepressor complex and an NRSF/REST-like factor, Chn, maintains inductive activity in developing photoreceptor cells by promoting Dl expression (Tsuda, 2006).

The corepressor complex that includes Ebi, SMRTER and Su(H) is required for expression of Dl in Drosophila photoreceptor cells. To identify genetic loci that are transcriptionally repressed by the Ebi corepressor, a screen was set up using an ectopic gene expression system (Gene Search System). Insertion of a Gene Search (GS) vector, a modified P-element carrying the Gal4 upstream activating sequence (UASG) near its 3' end, causes overexpression of a nearby gene under the control of the Gal4-UASG system. GS insertions into the chn locus were identified, whose overexpression phenotype in the eye using an eye-specific Gal4 driver (GMR-Gal4) was modified by reducing ebi activity. Thus the regulation of chn by Ebi-dependent transcriptional repression was studied (Tsuda, 2006).

In third instar larval-stage eye discs, the chn transcript is highly expressed in the morphogenetic furrow (MF), where photoreceptor differentiation initiates, but is downregulated in cells in the later stage photoreceptor development. In ebi mutant eye discs, however, chn expression becomes detectable in differentiating photoreceptor cells, and its expression in the MF is increased, suggesting that Su(H) in association with Ebi and SMRTER represses chn transcription in the eye disc (Tsuda, 2006).

To reveal the role of Su(H) as an activator, chn expression was examined when the level of Su(H) expression was reduced. Removing one copy of Su(H) suppresses the loss-of-Dl expression phenotype in ebi mutants. It was found that reducing one copy of Su(H) suppresses ectopic chn expression in ebi mutants, suggesting that ectopic expression of chn in ebi mutants is Su(H)-dependent. RT-PCR analysis of chn expression in ebi- eye discs differing in the dosage of Su(H) gene also supported these results. Strong reduction of Su(H) expression alone reduced expression of chn in the MF; this expression became weaker and was slightly broader. The phenotype of ebi, Su(H) double mutants is almost the same as Su(H) single mutants , suggesting that Su(H) acts as an activator in the absence of Ebi. This might be due to dual functions of Su(H) as an activator or repressor. Hence, reducing the amount of Ebi in the corepressor complex involving Su(H) might convert Su(H) to an activator by permitting the replacement of the corepressor complex with NICD (Tsuda, 2006).

To reveal the molecular nature of transcriptional regulation of chn by Su(H), Su(H) target sites were sought in the genomic region of chn. Since Su(H) binds slightly degenerate sequences, it was not easy to identify the functional Su(H) binding region from a simple genomic search. An alternative approach was taken to map the chn genomic region, which is regulated by Su(H) in the normal chromosomal context. Ebi-mediated repression involves SMRTER, a corepressor that recruits histone deacetylases and induces the formation of inactive chromatin, which spreads from the site where Su(H) recruits the corepressor complex. Promoters near the Su(H)-binding site are thus expected to be downregulated in an Ebi-dependent manner. Four insertion lines of the GS vector were identified in the chn promoter region. All these GS lines caused ectopic expression of chn with consequent abnormal eye morphology when they were crossed with GMR-Gal4. If the effect of the Ebi/SMRTER corepressor complex reaches the UASG in those insertions, reduction of Ebi activity will derepress UASG and further enhance activation by GMR-Gal4. One copy of a dominant-negative construct of ebi (GMR-ebiDN) caused only a mild defect in eye morphology and weak, if any, ectopic expression of chn. GMR-ebiDN strongly enhanced the overexpression phenotype of chnGS17605 and chnGS11450, which contained GS vector insertions (-474 and -734, respectively) upstream of the transcriptional start site. However, GMR-ebiDN failed to enhance the overexpression phenotype of other GS lines (chnGS2112 and chnGS17892) that were inserted downstream (+773 and +1040, respectively) of the first exon. From these results, it is concluded that Ebi-dependent transcriptional repression is targeted to the proximity of the transcriptional initiation site of the chn promoter (Tsuda, 2006).

Chn is a 1108-amino-acid protein with multiple C2H2-type zinc-finger motifs. Although no highly homologous gene within the mammalian genome could not be detected using BLAST, a small sequence of similarity between the N-terminal zinc-finger motif of Chn and the fifth zinc-finger of human NRSF/REST was found. Chn has several structural and functional similarities to human NRSF/REST, as follows. First, Chn and NRSF/REST each contain an N-terminal region with multiple zinc-finger motifs (five motifs in 264 residues in Chn and eight motifs in 251 residues in NRSF/REST), followed by a cluster of S/T-P motifs (serine or threonine followed by a proline) and a single zinc-finger motif at the C terminus. Second, the C-terminal region of NRSF/REST binds a corepressor, CoRest, which serves as an adaptor molecule to recruit a complex that imposes silencing activities. The Drosophila homolog of CoREST (dCoREST) (Andres, 1999; Dallman, 2004) can associate with the C-terminal half of Chn in cultured S2 cells. Finally, NRSF/REST binds to NRSE/RE1, a 21-bp sequence located in the promoter region of many types of neuron-restricted genes, via the N-terminal zinc-finger motifs. It was found that a recombinant protein containing the N-terminal zinc-fingers of Chn bound specifically to the NRSE/RE1 sequence in vitro. Thus, the structural similarity to NRSF/REST, binding to dCoREST and the DNA-binding specificity of Chn suggest that it is a candidate for a functional Drosophila homolog of NRSF/REST (Tsuda, 2006).

If Chn acts as a regulator of neural-related functions, as suggested for NRSF/REST, then Chn would be expected to bind to a regulatory region common to many types of neural-related genes in Drosophila. Numerous sequences similar to NRSE/RE1 were identified in the Drosophila genome, and their binding to Chn was assessed by EMSA. Using these sequences, a consensus binding sequence for Chn (Chn-binding element (CBE), 5'-BBHASMVMMVCNGACVKNNCC-3') was derived. 26 CBEs were identified within 10 kb of annotated genes from the Drosophila genome. Binding to Chn was confirmed for 18 CBEs using EMSA competition assay. Genes containing the CBE include dopamine receptor 2 (DopR2) and the potassium channel, ether-a-go-go, for which the mammalian homologs are target genes of NRSF/REST. These results suggest that the CBE is a good indicator of Chn binding sites and that Chn regulates many types of neural-related genes, as is implicated for NRSF/REST. However, it was found that divergent forms of CBE adjacent to hairy and extramacrochaetae were bound specifically by Chn. Likewise, some of the CBE sites failed to bind to Chn. Thus, a further refinement will be necessary to predict a definitive set of Chn binding sites in the Drosophila genome (Tsuda, 2006).

Although it has been established that mammalian NRSF/REST is a key regulator of neuron-specific genes, attempts to isolate invertebrate homolog of NRSF/REST have so far failed to identify a true homologous factor in invertebrates. The properties of Chn, including the similarity in DNA-binding specificity, association with CoREST and transcriptional repressor activity, suggest that Chn is a strong candidate for a functional Drosophila homolog of NRSF/REST. chn was originally identified by its requirement in the development of the PNS. This study identified a number of candidate target genes of Chn, a large fraction of which is implicated in neural function and/or gene expression. It is expected that further analysis of these candidates will provide valuable information about chn function in vivo, which may be extended to the understanding of NRSF/REST (Tsuda, 2006).

The Chn mutation blocks eye development by preventing the initiation of MF, a process requiring Notch signaling. This phenotype is likely owing to a loss of Notch function, because elevated Dl expression is known to block Notch signaling. The function of Chn during the early stage of eye development might be to regulate Notch signaling at an appropriate level by downregulating Dl. It is possible that Chn-mediated modulation places a variety of Notch functions in eye under the influence of EGFR signaling and provides flexibility in its regulation (Tsuda, 2006).

Although chn is expressed in the MF, genetic analyses show that small clones of chn mutant cells permit progression of the MF and photoreceptor differentiation. It is speculated that the repressive effect of Chn is overcome by other signals in the MF, such as hedgehog signaling, which strongly induces Dl (Tsuda, 2006).

Developing photoreceptor cells are exposed to the EGFR ligand, Spitz, and the Notch ligand, Dl, and each cell must assess the level of the two signals and respond appropriately to perform each task of photoreceptor cell specification and induction of non-neural cone cells. This question was investigated by studying the expression of Dl in photoreceptor cells. chn was identified as a direct target of Ebi/SMRTER-dependent transcriptional repression and as a repressor of Dl expression. The abrogated expression of Dl in ebi mutants was recovered by reducing one copy of chn, suggesting that the negative regulation of chn by ebi is indeed prerequisite for photoreceptor cell development (Tsuda, 2006).

Genetic data suggest that Su(H) may activate or repress chn expression. This idea is supported by data showing that Ebi/SMRTER and NICD are recruited to the promoter region of chn. The Ebi/SMRTER complex formed in this region did not contain any detectable level of the intracellular domain of Notch (NICD), suggesting that the binding of Ebi/SMRTER and NICD to this region may be mutually exclusive, and therefore it is expected that a regulatory system controls the balance between the active and repressive states of Su(H). Taken together, these results suggest that chn is a key factor in the crosstalk between two major signal transduction pathways: the EGFR-dependent pathway and the Notch/Delta-dependent pathway (Tsuda, 2006).

In the mammalian system, competition between SMRT and NICD for interaction with RBPJkappa determines the state of RBPJkappa-dependent transcriptional activity. Extracellular signaling may modulate this competition; diverse signaling pathways modulate the functions of N-CoR/SMRT. The current findings would prompt investigations of potential interaction of two repression systems of NRSF/REST and N-CoR/SMRT, and their regulation by Notch and EGF signaling in mammalian neuronal differentiation (Tsuda, 2006).

Regulation of Delta in trachea

The patterned branching in the Drosophila tracheal system is triggered by the FGF-like ligand Branchless (Bnl) that activates a receptor tyrosine kinase Breathless (Btl) and the MAP kinase pathway. A single fusion cell at the tip of each fusion branch expresses the zinc-finger gene escargot, leads branch migration in a stereotypical pattern and contacts with another fusion cell to mediate fusion of the branches. A high level of MAP kinase activation is also limited to the tips of the branches. Restriction of such cell specialization events to the tip is essential for tracheal tubulogenesis. Notch signaling plays crucial roles in the singling out process of the fusion cell. Notch is activated in tracheal cells by Branchless signaling through stimulation of Delta (Dl) expression at the tips of tracheal branches and activated Notch represses the fate of the fusion cell. In addition, Notch is required to restrict activation of MAP kinase to the tips of the branches, in part through the negative regulation of Branchless expression. Notch-mediated lateral inhibition in sending and receiving cells is thus essential to restrict the inductive influence of Branchless on the tracheal tubulogenesis (Ikeya, 1999).

Six primary branches form in the tracheal primordia, among which the dorsal branch (DB), anterior and posterior dorsal trunk (DTa, DTp), and anterior and posterior lateral trunk (LTa, LTp) migrate along a stereotyped path to be connected with other branches from adjacent primordia. These fusion branches are capped with fusion cells that express Esg. The remaining visceral branch (VB) migrates to reach the internal organs. Terminal cells expressing Drosophila serum response factor (DSRF) are formed in each primary branch except in DTs and later differentiate multiple tracheoles. High expression of DL mRNA and protein is expressed in the DT of stage-15 embryos. Cells in the tracheal primordium just after invagination expressed Dl uniformly. At early stage 11, Dl expression started to be elevated in 2-3 cells at the tip of the branches in which outgrowth had begun and the number of the Dl-expressing cells was reduced to one at late stage 11. At stage 14, high Dl expression remains only in the DT, which has completed fusion. Ser protein also accumulates at the apical side of the DT cells at the same stage. In the case of the trachea, the level of N protein expression remains uniform, suggesting that the expression level of Dl, or its potentiation, must be crucial for N activation. An esg-lacZ reporter is initially expressed in 2-3 cells at the tip of the fusion branches in mid stage 11 embryos, and is downregulated to be maintained in only a single cell at the tip of each fusion branch at late stage 11. These cells also express a high level of Dl. Therefore, localized elevation of Dl expression in stage 11 correlates well with the selection process of a single fusion competent cell (Ikeya, 1999).

When tracheal cells became unresponsive to Bnl due to btl mutation, no sign of primary branching and Dl upregulation is observed. On the contrary, when Btl is hyperactivated by overexpression of Bnl in all the tracheal cells, primary branching is severely inhibited and Dl expression is elevated. These results suggest that elevation of Dl expression is triggered by the external signal Bnl. The results also suggest that the N/Dl pathway may mediate the Bnl signal to control cell migration and cell fate decisions (Ikeya, 1999).

In Nts1 embryos grown under non-permissive condiditons a misrouting defect was observed in DB, which normally elongates to the dorsal midline where it meets its counterpart from the other side of the metamere. DBs are often curved in the anteroposterior direction and make contact with the tip of DB from the same side. The misrouted DBs accumulate a luminal component detectable by 2A12 antibody at the ectopic contact sites, but do not appear to fuse properly. Cell migration defect is also observed in DB. DB consists of a total of 5-7 cells in the case of Tr5, of which two specialized cells are located at the tip. One is the terminal cell, from which a thin terminal branch sprouts: the other is the fusion cell. The remaining stalk cells are located between the tip and DT at regular intervals. In Nts1 mutants, the number of cells at the tip is increased with a corresponding decrease in the number of stalk cells, the latter having become unusually elongated. The total number of cell nuclei do not change compared to the controls, so no additional mitosis occurs. This cell migration phenotype suggests that stalk cells and terminal cells acquire a property of fusion cells to become localized at the tip of DB. Since Esg represses DSRF expression and terminal branching, the loss of terminal branching in Nts1 embryos may be the consequence of ectopic Esg expression. Similar defects in Esg and DSRF expression are also observed in null N mutants. These results suggest that in N minus embryos, several terminal and stalk cells are recruited to the fate of fusion cells (Ikeya, 1999).

A three-step model is presented for tubule formation in fusion branches. During induction, exogenously supplied Bnl activates its receptor Btl in equivalent tracheal cells where N is inactive. The signal is transduced by activation of MAPK to stimulate Dl expression. The expression of Bnl is also regulated negatively by N signaling. During lateral inhibition, induced Dl activates N in neighboring cells, which in turn, represses esg transcription. N may also repress Dl expression. However, a high level of Dl inhibits N signaling in a cell autonomous manner, allowing activation of esg and MAPK. A small difference in the response to Bnl within equivalent tracheal cells is amplified to select out a single fusion cell with a high level of Dl and esg expression. During tubule formation, the fusion cell becomes the only cell that responds to Bnl and becomes motile. Maintenance of Btl activity by Bnl would limit the migration toward the source of Bnl. Other tracheal cells follow fusion cells to become stalk cells (Ikeya, 1999).

Decapentaplegic (Dpp) signaling determines the number of cells that migrate dorsally to form the dorsal primary branch during tracheal development. Dpp is expressed in dorsolateral epidermal clusters located near the tips of the outgrowing dorsal branches. The Dpp receptor, Thick veins, is expressed in all tracheal cells during embryogenesis and is required for in dorsal branch outgrowth ectopic activation of fusion markers in cells of the dorsal branch. Dpp signaling is required for the differentiation of one of three different cell types in the dorsal branches, the fusion cell. In Mad mutant embryos or in embryos expressing dominant negative constructs of the two type I Dpp receptors in the trachea the number of cells expressing fusion cell-specific marker genes is reduced and fusion of the dorsal branches is defective. Ectopic expression of Dpp or the activated form of the Dpp receptor Tkv in all tracheal cells induces ectopic fusions of the tracheal lumen and ectopic expression of fusion gene markers in all tracheal branches. Delta is among the fusion marker genes that are activated in the trachea in response to ectopic Dpp signaling. In conditional Notch loss of function mutants, additional tracheal cells adopt the fusion cell fate. Ectopic expression of an activated form of the Notch receptor in fusion cells results in suppression of fusion cell markers and disruption of the branch fusion. The number of cells that express the fusion cell markers in response to ectopic Dpp signaling is increased in Notch ts1 mutants, suggesting that the two signaling pathways have opposing effects in the selection of the fusion cells in the dorsal branches (Steneberg, 1999).

Several alleles of Delta were identified using an enhancer trap screen for genes that are selectively expressed in the tracheal fusion cells. In these strains, the Delta-lacZ marker is initially expressed homogeneously in all tracheal cells, but becomes up-regulated in the fusion cell from embryonic stage 13, approximately at the same time that the first fusion marker genes initiate their expression in the fusion cell. Inactivation of Notch signaling by a conditional allele of the receptor results in more cells acquiring the fusion cell fate. Delta-lacZ is also up-regulated in the extra fusion cells, indicating that Notch signaling in the trachea also regulates the expression of Delta, as has been suggested for other Notch signaling events. In Notchts1 mutants the total number of cells in the dorsal branch remains the same; the number of cells expressing the terminal cell marker is not decreased. Thus, Notch is required for the suppression of the fusion cell program in the three cells that will remain in the stalk of the branch. Accordingly, expression of an activated form of Notch in all tracheal cells, or in the fusion cells only, inhibits branch fusion and suppresses the expression of fusion marker genes. Cell counts in these affected dorsal branches show that the number of cells is unchanged; this suggests that the presumptive fusion cell does not undergo apoptosis but instead remains in the stalk of the branch. However, in embryos expressing the activated form of Notch in all trachea, a few additional cells express the terminal marker genes and sprout off the dorsal branches, but this is likely due to the inability of the presumptive fusion cell to produce the Hdc inhibitor of tracheal cell branching (Steneberg, 1999).

The localized expression of Dpp in the dorsal epidermis is required for the induction of the fusion cell fate, whereas Notch signaling has the opposite effect in the expression of fusion marker genes. Since the number of cells that acquire the fusion cell fate in response to Dpp becomes higher when the amount of functional Notch is reduced, it has been proposed that Notch signaling in the trachea is required for the selection of a single fusion cell among the group of cells that receive the Dpp signal at the tip of the dorsal branch. Furthermore, Dpp signaling can activate expression of Delta suggesting that Delta expression in the fusion cell may be directly activated by downstream effectors of Dpp signaling. Such transcription factors include Mad, Knirps and Knirps-like, all of which act downstream of Dpp signaling during primary branching. Alternatively, the activation of Delta in the cells in the stalk of the branch may be suppressed by negative regulators of Dpp signaling (such as Daughters against dpp (Dad) or Brinker, both of which are regulated by Dpp signaling). The requirement for Dpp signaling in branch fusion does not seem to be a general theme for all tracheal branches. Dpp is not expressed in the proximity of the dorsal trunk fusion cells and dorsal trunk fusion is not affected in embryos mutant for Mad, tkv or in embryos expressing the dominant negative forms of the receptors in all tracheal cells. These results argue that the induction of fusion cell fate in different branches employs different signaling mechanisms. Such a signaling event that may be responsible for the activation of fusion genes in the dorsal trunk may be provided by the Spi/EGFR pathway. Mutations in members of this pathway are defective in dorsal trunk formation, and in the developing Drosophila wing; rhomboid, a component of EGF signaling, has been shown to regulate the expression of Delta. In contrast to the local requirement of Dpp in the dorsal branch, Notch signaling is a prerequisite for the selection of the fusion cell in all primary branches that will extend a fusion sprout. Thus, even though the inductive signals required for the expression of the fusion cell-specific marker genes may be different, they all appear to result in the up-regulation of Delta; they signal through Notch to select a single cell at tip of the branch that will execute the complex cellular dynamics of branch fusion (Steneberg, 1999).

The tubular epithelium of the Drosophila tracheal system forms a network with a stereotyped pattern consisting of cells and branches with distinct identity. The tracheal primordium undergoes primary branching induced by the FGF homolog Branchless; it differentiates cells with specialized functions such as fusion cells, which perform target recognition and adhesion during branch fusion, and extends branches toward specific targets. Specification of a unique identity for each primary branch is essential for directed migration, because a defect in either the Egfr or the Dpp pathway leads to a loss of branch identity and the misguidance of tracheal cell migration. The role of Wingless signaling in the specification of cell and branch identity in the tracheal system has been investigated. Wingless and its intracellular signal transducer, Armadillo, have multiple functions, including specifying the dorsal trunk through activation of Spalt expression and inducing differentiation of fusion cells in all fusion branches. Moreover, Wingless signaling regulates Notch signaling by stimulating Delta expression at the tip of primary branches. These activities of Wingless signaling together specify the shape of the dorsal trunk and other fusion branches (Chihara, 2000).

During primary branching, Delta protein accumulates at the tips of primary branches, restricting the differentiation of excess fusion cells by stimulating Notch signaling. Wg signaling is required for localized Delta expression. Ectopic expression of an activated form of Arm in all tracheal cells can activate Delta as well as Delta-lacZ expression, suggesting that Wg signaling stimulates Delta expression at the transcriptional level. A similar conclusion has been drawn from studies of Wg function in dorsoventral patterning of Wing imaginal discs. Another mechanism whereby Wg signaling interacts with Notch has been proposed. Dishevelled (Dsh), which is a transducer of Wg signaling acting upstream of Arm, inhibits Notch activity in Drosophila wing discs and interacts with the intracellular domain of Notch in yeast cells. This mechanism is distinct from the proposed mechanism of Notch inhibition by Wg signaling, since activated Arm acting downstream of Dsh causes a phenotype of Notch inhibition. These mechanisms are not mutually exclusive, however, and may reflect a different aspect of complex self- and cross-regulatory interactions of the two signaling pathways (Chihara, 2000).

How does the localized Delta induced by Wg signaling act in tracheal cells? As revealed by the study on Drosophila wing disc development, Notch ligands have a cell-autonomous dominant-negative effect on Notch activity in addition to the well-established role of lateral inhibition of cell differentiation. Clones of cells lacking both Delta and Serrate show a sign of Notch hyperactivation and clones of cells expressing high level of Delta autonomously inhibit Notch target genes. The same relationship between Notch and Delta appears to exist in the trachea, since overexpression of Delta shows phenotypes similar to loss of Notch function. Since Esg is expressed in cells with the highest Delta expression in primary branches and is normally inhibited by Notch signaling, it was proposed that Delta-dependent inhibition of Notch provides permissive conditions for fusion cell differentiation. Thus regulation of Delta by Wg signaling is an important mechanism of fusion cell-fate determination (Chihara, 2000).

Delta expression in tracheal cells is also under the influence of Bnl/Btl signaling. Loss of Btl causes a reduction of Delta and overexpression of Bnl leads to excess Delta expression. These observations suggest that the localized expression of Delta in the developing trachea requires both Wg and Bnl signaling, implying that the two signals synergistically stimulate Delta expression. It is proposed that the two diffusible ligands Bnl and Wg, expressed in distinct special domains, separately exert an inductive influence on the tracheal primordium. Delta integrates the two inductive signals and elevates its expression in sharply defined regions at the tip of the primary branches, and initiates the cell-fate restriction program. This mechanism is likely to be useful for sharpening the response of cells to multiple diffusible ligands (Chihara, 2000).

Wg signaling controls the formation of DT by regulating at least three target genes (sal, esg and Delta) in distinct ways. Sal is expressed in all DT cells and is required for directed migration along the anterior and posterior directions. Most of the cells in the Egfr domain can respond to Wg signaling by expressing Sal, and the expression of Sal is not affected by excess Delta. It is proposed that Sal expression is regulated by Wg signaling but not by Notch signaling, and that it serves as a major mediator of Wg signaling in determining DT identity. Regulation of Esg is more complex. Although Esg expression is stimulated by Wg signaling, it is normally limited to a single cell on each branch due to repression by Notch. Wg signaling activates Esg expression independently of Delta. It is proposed that Wg signaling bifurcates after activation of Arm, activating Esg on the one hand, and Delta on the other. Elevated Delta activates Notch in nearby cells, leading to repression of Esg in the stalk of tracheal branches. These combinatorial effects limit Esg expression to the tip of fusion branches. Stimulation of both positive and negative regulation of Esg by a single inductive signal comprises a self-limiting process of cell-fate determination and accounts for the assignment of single fusion cells that mark the end of the tracheal tubule. In combination with the specification of thick tubules through regulation of Sal, Wg signaling determines the shape of the tracheal tubule (Chihara, 2000).

Regulation of Delta in the leg

Segmentation is a developmental mechanism that subdivides a tissue into repeating functional units, which can then be further elaborated upon during development. In contrast to embryonic segmentation, Drosophila leg segmentation occurs in a tissue that is rapidly growing in size and thus segmentation must be coordinated with tissue growth. Segmentation of the Drosophila leg, as assayed by expression of the key regulators of segmentation, the Notch ligands and fringe, occurs progressively and this study defines the sequence in which the initial segmental subdivisions arise. The proximal-distal patterning genes homothorax and dachshund are positively required, while Distal-less is unexpectedly negatively required, to establish the segmental pattern of Notch ligand and fringe expression. Two Serrate enhancers that respond to regulation by dachshund are also identified. Together, these studies provide evidence that distinct combinations of the proximal-distal patterning genes independently regulate each segmental ring of Notch ligand and fringe expression and that this regulation occurs through distinct enhancers. These studies thus provide a molecular framework for understanding how segmentation during tissue growth is accomplished (Rauskolb, 2001).

In recent years the key role of the Notch signaling pathway in the segmentation and growth of the Drosophila leg has been established. Notch signaling must be localized within each leg segment to promote the formation of boundaries (joints) that separate each leg segment and to induce leg growth. This requirement for a segmentally repeated pattern of Notch activation is accomplished by restricting the expression of the regulators of Notch activation, Serrate, Delta and fringe, to one ring per segment. By examining the expression of the Notch ligands and fringe during leg development, it has been possible to determine the progressive order in which leg segmentation is established. At early third instar, a single ring of Serrate, Delta and fringe expression is present within the coxa. The next ring to arise is located within the presumptive femur. At mid third instar, expression arises within presumptive tarsal segments 2 and 5. Subsequent expression is observed in the tibia and more tarsal segments, such that ultimately, by the end of third instar, a ring of expression is present in each presumptive leg segment, adjacent to each prospective leg segment border. Thus, segmentation of the Drosophila leg occurs progressively and in a reproducible pattern (Rauskolb, 2001).

Previous studies investigating the expression of a reporter gene [E(spl)mß-CD2] regulated downstream of Notch activation led to the conclusion that the first segment boundary to form was between tarsal segments 4 and 5. Additional rings of expression were then observed in the tarsus and then eventually in all leg segments. This led to the suggestion that the first segmental boundaries to form correspond to the most distal segments. However, further examination of this reporter gene indicates that expression is observed in proximal cells prior to expression within the tarsus. Moreover, temperature shifts of a conditional Notch allele at different stages of development demonstrate that the temperature-sensitive period for Notch in proximal segmentation occurs before that in tarsal segmentation. The conclusion is reached that leg segmentation does not occur in a simple distal to proximal order, nor proximal to distal order, nor are the most proximal and distal segments established first and other segments added by intercalation. Rather, the establishment of Drosophila leg segmentation occurs in a complex sequence (Rauskolb, 2001).

A general theme in patterning during development is the subdivision of tissues initially by genes expressed in broad, partially overlapping domains, which through combinatorial control, subsequently regulate the expression of downstream genes to generate a repeating pattern. The studies presented here demonstrate that leg segmentation follows this same theme. The 'leg gap genes' Hth, Dac, and Distal-less are expressed in broad domains in the leg disc that encompass more than a single segment. Initially expression of these genes is largely nonoverlapping, but as the leg disc grows, the expression patterns of the leg gap genes change such that five different domains of gene expression are established. The analysis of the regulation of Notch ligand and fringe expression during leg development reveals two fundamental aspects of leg development. (1) These leg gap genes are key components in regulating the expression of the molecules controlling segmentation. Indeed, the effect of these leg gap genes on leg segmentation and growth can be accounted for by their regulation of Serrate, Delta and fringe expression. (2) The expression of each ring of Serrate, Delta and fringe is controlled by its own unique combination of regulators, apparently acting through independent enhancers (Rauskolb, 2001).

How do these three transcription factors regulate the formation of nine segments? Since the requirements for and the expression of the leg gap genes encompasses all leg segments, it is unlikely that there are additional leg gap genes yet to be identified. Rather, a collection of distinct combinatorial approaches is used to establish a segmental pattern of Serrate, Delta and fringe expression (Rauskolb, 2001).

In early third instar leg discs, there are two domains of gene expression: proximal cells express Hth and distal cells express Distal-less. Hth autonomously promotes the expression of Serrate, while Distal-less may prevent expression more distally, giving rise to a ring of expression in the coxa. Additionally, Distal-less-expressing cells may signal to the Hth-expressing cells to restrict Serrate expression to the distal edge of the Hth domain. As the leg disc grows, cells in an intermediate position, lying between the Hth and Distal-less domains, begin to express Dac. Dac, as shown in this study, is both necessary and sufficient to induce the expression of Serrate, Delta and fringe within the femur. Since they are not expressed in all Dac-expressing cells, other factors appear to be required to promote their expression in the proximal femur. The nonautonomous induction of Serrate expression by Hth suggests that this may be accomplished by a signal (X) emanating from the neighboring Hth-expressing cells. By mid third instar stages, expression of Serrate, Delta and fringe is also observed in tarsal segments 2 and 5, within cells expressing Distal-less but not Dac. Given that Distal-less is necessary and sufficient to repress their expression, Serrate, Delta and fringe expression within the tarsus appears to be induced by a mechanism that overrides the repressive effects of Distal-less. Subsequently, expression of Serrate, Delta and fringe is observed within the tibia, in cells expressing both Dac and Distal-less. Dac is necessary for expression of Serrate within the tibia, and its role here may be to overcome the repressive effects of Distal-less. It is also worth noting that the tibia ring of expression is not established at the time when cells first express both Dac and Distal-less. This may be because Dac levels may not be sufficiently high enough to overcome the repression by Distal-less. Clearly levels of Dac expression are critical because simply increasing Dac levels is sufficient to promote Serrate expression in cells already expressing endogenous levels of Dac. This observation can be explained if high levels of Dac expression in cells already expressing Dac override the function of inhibitory regulators of Serrate expression, such as Distal-less, where the expression of these genes overlap. Although late stages of leg segmentation were not investigated in this study, it has been noted that Hth, Dac and Distal-less are co-expressed in the presumptive trochanter late in leg development. It is thus hypothesized that Serrate, Delta and fringe expression is established by the combined activities of the three leg gap genes in the trochanter (Rauskolb, 2001).

Although these here have focused on the regulation of Serrate expression, it is thought that not only Serrate, but also Delta and fringe, receive primary regulatory input from the leg gap genes. Delta and fringe expression, like Serrate, is positively regulated by Dac. Moreover, Dl and fringe mutants have stronger leg segmentation phenotypes than Ser mutants, and thus Delta and fringe expression cannot simply be regulated downstream of Ser. The identification of two separate Ser enhancers, directing expression in the proximal versus distal leg, argues against Serrate being regulated downstream of Dl and fringe. Thus, the simplest model is that expression of all three genes is regulated directly by the leg gap genes. The regulation of Serrate, Delta and fringe expression in each segment appears to occur through independent and separable enhancer elements, supported by the analysis of the Ser reporter genes. This is reminiscent of what occurs during Drosophila embryonic segmentation, where separable enhancer elements direct different stripes of pair-rule gene expression (Rauskolb, 2001).

Importantly, Notch signaling may actually coordinate progressive segmentation of the leg with leg growth. For example, in early leg discs there is a single ring of Serrate expression within the coxa, in Hth-expressing cells immediately adjacent to Dac-expressing cells. However, by the time the femur ring arises, the coxa ring of Serrate expression has been displaced and is no longer within cells immediately adjacent to the Dac-expressing cells; rather, there are Hth-expressing cells lying in between that do not express Serrate. Thus, it is postulated that once Serrate, Delta and fringe expression is established within the coxa, Notch is activated, which promotes local cell proliferation, thereby displacing the coxa ring. This then allows for the femur ring of expression to be established in cells that are not immediately adjacent to the coxa expression ring. This mechanism also requires that once a ring of ligand expression is established in a particular segment, this expression must be maintained such that it is not influenced by later alterations in relation to leg gap gene expression. This maintenance could be accomplished by a feedback loop between Notch activation and ligand expression, similar to what has been observed during late wing development, where Notch activation cell autonomously represses ligand expression and nonautonomously induces ligand expression in flanking cells by regulating the expression of a signaling molecule. Preliminary studies have indicated that Notch activation can influence Notch ligand expression in the developing leg (Rauskolb, 2001).

The distal region of the Drosophila leg, the tarsus, is divided into five segments (ta I-V) and terminates in the pretarsus, which is characterized by a pair of claws. Several homeobox genes are expressed in distinct regions of the tarsus, including aristaless (al) and lim1 in the pretarsus, Bar (B) in ta IV and V, and apterous (ap) in ta IV. This pattern is governed by regulatory interactions between these genes; for example, Al and Bar are mutually antagonistic, resulting in exclusion of Bar expression from the pretarsus. Although Al is necessary, it is not sufficient to repress Bar, indicating another factor is required. This factor has been identified as the product of the C15 gene, also termed clawless, a homeodomain protein that is a homolog of the human Hox11 oncogene. C15 is expressed in the same cells as al -- together, C15 and Al appear to directly repress Bar and possibly to activate Lim1. C15/Al also act indirectly to repress ap in ta V, i.e., in surrounding cells. To do this, C15/Al autonomously repress expression of the gene encoding the Notch ligand Delta (Dl) in the pretarsus, restricting Dl to ta V and creating a Dl+/Dl− border at the interface between ta V and the pretarsus. This results in upregulation of Notch signaling, which induces expression of the bowl gene, the product of which represses ap. Similar to aristaless, the maximal expression of C15 requires Lim1 and its co-factor, Chip. Bar attenuates aristaless and C15 expression through Lim1 repression. Aristaless and C15 proteins form a complex capable of binding to specific DNA targets, which cannot be well recognized solely by Aristaless or Clawless (Campbell, 2005; Kojima, 2005).

If Notch signaling induces bowl expression and C15 is also required for bowl expression, it was predicted that C15 upregulates Notch signaling by regulating the expression of the Notch ligand responsible for activation of bowl. Although, both Notch ligands, Delta (Dl) and Serrate are expressed in leg discs, it was discovered that only Dl is required to induce expression of bowl. bowl expression is lost in homozygous Dl mutant clones, although, if positioned appropriately, wild-type cells can rescue bowl expression in adjacent cells laterally and distally. Curiously, nub, which was also thought to be a target of Notch signaling, is still expressed in Dl mutant cells (even far from wild-type cells), albeit in an irregular pattern (it is expressed at normal levels in some cells, but at lower levels or not at all in others. Misexpression of Dl can also induce ectopic bowl expression both in adjacent cells and in the cells misexpressing Dl. However, in large clones, cells in the center of the clone do not express bowl, which is only expressed in the cells at the edge of the clone and in the cells immediately adjacent to the clone. Nub is not ectopically expressed following misexpression of Dl (Campbell, 2005).

In wild-type mid-third instar discs, Dl expression is upregulated in ta V, overlapping with Nub, but not with C15. Distally, it overlaps partially with Bowl, although Bowl is also expressed even more distally. Proximally, however, Dl does not appear to induce expression of Bowl, suggesting there is a repressor of Bowl expressed in this location. This is supported by the inability of cells misexpressing Dl in this position (proximal ta V, ta IV) to activate Bowl. Although it might be predicted that C15 would induce expression of Dl, in fact the opposite was found, and C15 actually represses Dl in the center of the disc. In C15 mutants, Dl expression expands into the center of the disc and misexpression of C15 can repress expression of Dl. How C15-repression of Dl can result in upregulation of Notch signaling in cells in ta V surrounding the pretarsus is discussed below (Campbell, 2005).

dAP-2 and defective proventriculus regulate Serrate and Delta expression in the tarsus of Drosophila melanogaster

The segmentation of the proximal-distal axis of the Drosophila leg depends on the localized activation of the Notch receptor. The expression of the Notch ligand genes Serrate and Delta in concentric, segmental rings results in the localized activation of Notch, which induces joint formation and is required for the growth of leg segments. This study reports that the expression of Serrate and Delta in the leg is regulated by the transcription factor genes dAP-2 and defective proventriculus. Previous studies have shown that Notch activation induces dAP-2 in cells distal and adjacent to the Serrate/Delta domain of expression. Serrate and Delta are ectopically expressed in dAP-2 mutant legs, and Serrate and Delta are repressed by ectopic expression of dAP-2. Furthermore, Serrate is induced cell-autonomously in dAP-2 mutant clones in many regions of the leg. It was also found that the expression of a defective proventriculus reporter overlaps with dAP-2 expression and is complementary to Serrate expression in the tarsal segments. Ectopic expression of defective proventriculus is sufficient to block joint formation and Serrate and Delta expression. Loss of defective proventriculus results in localized, ectopic Serrate expression and the formation of ectopic joints with reversed polarity. Thus, in tarsal segments, dAP-2 and defective proventriculus are necessary for the correct proximal and distal boundaries of Serrate expression and repression of Serrate by defective proventriculus contributes to tarsal segment asymmetry. The repression of the Notch ligand genes Serrate and Delta by the Notch target gene dAP-2 may be a pattern-refining mechanism similar to those acting in embryonic segmentation and compartment boundary formation (Ciechanska, 2007).

Sex combs reduced and Ultrabithorax establish differential expression of the proneural gene achaete by modifying expression of the achaete prepattern regulator Delta in Drosophila legs

Many studies have shown that morphological diversity among homologous animal structures is generated by the homeotic (Hox) genes. However, the mechanisms through which Hox genes specify particular morphological features are not fully understood. This issue was addressed by investigating how diverse sensory organ patterns are formed among the legs of the Drosophila adult. The Drosophila adult has one pair of legs on each of its three thoracic segments (the T1-T3 segments). Although homologous, legs from different segments have distinct morphological features. Focus was placed is on the formation of diverse patterns of small mechanosensory bristles or microchaetae (mCs) among the legs. On T2 legs, the mCs are organized into a series of longitudinal rows (L-rows) precisely positioned along the leg circumference. The L-rows are observed on all three pairs of legs, but additional and novel pattern elements are found on T1 and T3 legs. For example, at specific positions on T1 and T3 legs, some mCs are organized into transverse rows (T-rows). The T-rows on T1 and T3 legs are established as a result of Hox gene modulation of the pathway for patterning the L-row mC bristles. The findings suggest that the Hox genes, Sex combs reduced (Scr) and Ultrabithorax (Ubx), establish differential expression of the proneural gene achaete (ac) by modifying expression of the ac prepattern regulator, Delta (Dl), in T1 and T3 legs, respectively. This study identifies Dl as a potential link between Hox genes and the sensory organ patterning hierarchy, providing insight into the connection between Hox gene function and the formation of specific morphological features (Shroff, 2007).

It is proposed that T-rows are formed on T1 and T3 legs in response to Scr or Ubx alteration of the L-row prepattern via repression of Dl expression. Dl is expressed in narrow longitudinal stripes that correspond to the L-row primordia. Dl-expressing cells in the L-row primordia signal to adjacent cells to activate N signaling and repress ac expression in the hairy-OFF interstripes and in one hairy-ON interstripe, between the L-row 1 and 8 proneural fields. It is suggested that in T1 and T3 legs, reduction of Dl expression in cells with high-levels of Scr or Ubx establishes a zone where there is no repressive influence on ac expression, resulting in expression of Ac in broad domains from which the T-row precursors will be selected. Cells in the center of T-row primordia are presumably out of range of the Dl signaling that takes place at the interface of Dl-expressing and Dl-non-expressing cells. The anterior and posterior boundaries of Ac expression in the T-row primordia of T1 prepupal legs are likely established by Dl/N signaling. In T3 legs, in contrast, it appears that Hairy rather than Dl/N signaling establishes the boundaries of ac on either side of the T-row primordia. Reduced Dl expression in the T-row primordia of T3 legs, however, is likely required to establish a broader domain of Ac expression than would be observed in the corresponding domain of T2 legs (Shroff, 2007).

A key feature of the model for mC patterning is that position-specific expression of ac expression in the mC proneural fields is established mainly by repression and that differential mC patterns are generated by altering expression or function of the repressive factors, Hairy and/or Dl. It is suggested that altered Dl expression is required in order to reduce N signaling, which allows proneural gene expression within the T-row primordia. An alternative hypothesis is that Dl function in during leg mC development is limited to selection of SOPs via lateral inhibition and that regulation of Dl by Scr/Ubx alters lateral inhibition within the T-row proneural fields. However, the hypothesis that Scr/Ubx regulation of Dl alters the proneural prepattern is supported by several observations. A prepattern function for Dl in mC patterning has been previously demonstrated in the notum. Similarly, it has been observed that in prepupal legs with reduced Dl function, proneural Ac expression expands along the leg circumference and is excluded only from Hairy-expressing cells. Furthermore, in prepupal legs, proneural Ac expression fills the center of large clones lacking Dl function. It is also observed that N signaling is activated only in narrow stripes on either side, but not within the T-row proneural fields. The genetic observations are substantiated by analysis of an enhancer that directs ac expression in both the L-row and T-row proneural fields. This enhancer consists of an activation element that directs uniform expression of ac along the leg circumference and two associated repression elements, one that is N-responsive and another that is Hairy-responsive. This is consistent with genetic studies suggesting that in the absence of repressive influences from Hairy and Dl, proneural ac expression would be uniformly along the leg circumference. Combined, these observations suggest that the mC patterning pathway is modified upstream of proneural gene expression by establishment of differential Dl expression in legs from different thoracic segments (Shroff, 2007).

The finding that Dl expression is down-regulated in the T-row primordia, does not necessarily imply that Dl expression is incompatible with mC formation. That this is not the case is suggested by the observation that Dl is expressed in the L-row primordia. Previous studies in a number of tissues have shown that high-level N-ligand expression renders cells non-responsive to N signaling. Hence, it appears that Dl/N signaling at the boundary of Dl-expressing and Dl-non-expressing cells, not Dl expression per se, is incompatible with mC development (Shroff, 2007).

Many studies have made clear the importance of establishing spatially defined proneural gene expression, largely via transcriptional regulation, for patterning of both the vertebrate and invertebrate nervous system. For example, it has been shown that ectopic proneural gene expression causes disruption of the sense organ pattern in adults. In the leg, compromised hairy function results in ectopic proneural ac expression and disorganization of the adult mC pattern, including formation of extranumerary mCs. However, other studies have implicated post-transcriptional regulation of proneural gene function in neural patterning. This was suggested by a study that showed that generalized and transient sc expression in a background devoid of ac and sc function results in an almost normal sense organ pattern in adult flies. Studies in the notum have provided an explanation for this observation by identification of the Extra macrochaetae (Emc) protein as a post-transcriptional regulator of proneural gene function. Emc, an HLH protein that lacks a basic DNA binding domain, binds proneural bHLH proteins, such as Ac, and inhibits their activity. In the notum, emc is expressed in a complex pattern that partially overlaps proneural gene expression, and it appears that SOPs are selected from cells with the lowest levels of Emc. This would suggest that on the notum, competence to acquire a neural fate depends on the balance of proneural protein to Emc levels. It is probable that similar mechanisms function in leg mC patterning as well, since largely normal sense organ patterns are found in legs ubiquitously expressing Sc. These observations indicate that sense organ patterning is a complex process that involves regulation of both proneural gene expression and function. Hence, it would be of interest to assess the relative contribution of post-transcriptional regulation of proneural gene function on leg mC patterning (Shroff, 2007).

It is proposed that T-row mCs are selected from domains of up-regulated Scr or Ubx expression and that one essential function for Scr and Ubx in T-row development is repression of Dl expression. This proposal is supported by several lines of evidence. The requirement of Scr and Ubx in T-row formation was suggested by prior reports that loss of Scr or Ubx function results in transformation of T1 or T3 legs, respectively, toward a T2 fate. This study shows that adult legs heterozygous for reduced function alleles of Scr (ScrEdK6/ScrEfW22) exhibit almost complete loss of T-rows in the adult. Moreover, ectopic expression of Scr or Ubx induces T-row formation on T2 legs, on which T-rows are never normally observed. The domains of elevated Scr and Ubx expression in T1 and T3 prepupal legs correspond to the respective positions of T-rows in adult T1 and T3 legs. Furthermore, comparison of Scr expression to that of an SOP marker, sca-Gal4, shows that T-row mCs are selected from groups of cells that express high-level Scr on T1 legs (Shroff, 2007).

Strong evidence is provided that Scr and Ubx repress Dl expression in the T-row primordia. First, a correlation is observed between up-regulated Scr and Ubx expression and domains of reduced Dl expression. Second, loss and gain of function studies indicate that Scr and Ubx negatively regulate Dl expression. In ScrEdK6/ScrEfW22, prepupal legs, Dl is expressed in two longitudinal stripes overlapping the region of high-level Scr expression, whereas in wild type legs Dl stripes flank but do not overlap this domain. In addition, Dl is ectopically expressed in either Scr or Ubx loss of function clones within the T-row primordia. Consistent with loss of function results, it was found that ectopic high-level expression of Scr or Ubx results in repression of Dl expression (Shroff, 2007).

Taken together, these observations suggest a function for Scr and Ubx in specification of a T-row fate. However, the finding that the formation of T-rows in response to ectopic Scr or Ubx is confined to ventro-lateral regions along the circumference implies that there may be other positional cues, in addition to elevated Scr or Ubx expression, that are required for T-row specification. Hence, it is plausible that these genes function combinatorially with other factors to induce T-row formation. Wg, for example, is a good candidate since it is expressed in ventro-lateral regions of the leg. In addition, ectopic Wg expression results in expansion of T-row bristles in T1 legs. It is also plausible that, in addition to or instead of T-row promoting factors in ventro-lateral leg regions, there are factors outside these domains that inhibit T-row formation (Shroff, 2007).

These studies have elucidated a general pathway for leg mC patterning in which an early event is establishment of position-specific expression of the prepattern genes hairy and Delta, presumably in response to the global regulators of limb patterning. The spatial regulation of hairy expression during leg development has been investigated ant it has been determined that hairy expression is controlled by modular enhancer elements that integrate patterning information provided by the signaling molecules known to pattern the leg along its circumference, Hedgehog (Hh), Decapentaplegic (Dpp) and Wingless (Wg). Periodic Dl expression is partially established by Hairy. However, it is likely that Dl expression in the mC proneural fields is also regulated, like hairy, by genes that pattern the leg along the circumference (circumferential patterning genes), such as hh, dpp and wg (Shroff, 2007).

Up-regulated expression of Scr and Ubx at specific positions along the circumference and P/D axis of T1 and T3 prepupal legs is key to generating the T-row pattern, raising the question of how Scr and Ubx expression in the T-row primordia is regulated. It is proposed that Scr and Ubx expression is controlled by the circumferential patterning genes and genes that pattern the leg along the P/D axis (P/D patterning genes). For example, Scr and/or Ubx expression might be regulated by Wg, which is known to specify ventral leg identity and patterns the ventral leg along the A/P axis in a concentration dependent manner. Along the P/D axis, a number of genes, such as Distal-less and dachshund, are expressed in defined and partially overlapping domains and might function to define the extent of up-regulated Scr and/or Ubx expression. This model would suggest that circumferential and P/D patterning information is integrated by Scr and Ubx, implying that these Hox genes link the global regulators of leg development to local acting genes, such as ac, that specify a neural fate (Shroff, 2007).

These studies indicate that Scr and Ubx function early in T-row development to repress Dl expression, which allows formation of the T-row proneural fields. It will be of interest to determine whether Dl is a direct target of these Hox genes, especially since few direct Hox-gene targets have been identified to date. However, it is probable that Scr and Ubx have other functions in T-row development. Establishment of ac expression in the T-row primordia is an early and essential step of T-row development, but, while ac specifies a neural fate, it does not specify sensory organ type. Hence, it is likely that Scr and Ubx function, in conjunction with other factors, to specify 'T-row-type' mCs. For example, since the T-row mCs are less pigmented than L-row mCs, a potential role of Scr and Ubx in T-row development is to regulate genes involved bristle pigmentation. A second potential function for these Hox genes is in controlling growth in the regions of the legs where T-rows are formed. In T1 legs, for example, the region between L-rows 7 and 8, in which the T-rows are found, is larger than the corresponding region on T2 legs, implying that there is additional growth in this domain. Inconsistent with this hypothesis, however, is the observation that posterior compartment clones lacking Ubx function in the T3 basitarsus did not have a significant effect on basitarsal width (Shroff, 2007).

Another potential role for Scr and Ubx in patterning T-row bristles is to implement a mechanism for selection and organization of T-row mCs into transverse rows. The mechanisms through which the L-row and T-row bristles are selected and organized within their respective proneural fields are likely to differ substantially. The regular spacing of L-row mCs suggests that the L-row SOPs send inhibitory signals in all directions to establish their proper spacing. This is also suggested by the observation that in hairy mutant legs, Ac is expressed in four broad domains, similar to the broad T-row proneural fields, and the supernumerary mCs that are formed on hairy mutant legs are well spaced along the leg circumference. This would suggest that the lateral inhibitory signals are sent along both the leg circumference and P/D axis. Unlike the L-row bristles, the T-rows mCs are positioned directly adjacent to one another in straight regularly spaced transverse rows. How the T-row precursors are selected from a broad field of ac-expressing cells and are arranged in tandem in straight rows is not understood. Previous studies have implicated N and EGFR signaling in formation of organized T-rows. Although the current studies indicate that N-signaling is down-regulated in the T-row primordia, it is conceivable that N functions at later stages of T-row development to pattern the T-row bristles. For example, N might function to set the register and spacing of the T-rows. Also of interest is how the T-rows are aligned in tandem within the rows. It has been suggested that homophilic adhesion between mC SOPs might be involved in organizing T-row bristles. Hence, it is plausible that Hox genes regulate expression of genes involved in adhesion, N signaling and/or EGFR signaling. Investigation of the mechanisms of T-row SOP selection and organization will provide an opportunity to uncover a potential connection between Hox gene function and morphogenesis (Shroff, 2007).

The proposed function for Scr and Ubx in T-row patterning bears some similarity to that described for Ubx in generation of diverse trichome patterns among the T2 legs of various Drosophila species. It has been shown that late pupal expression of Ubx in the T2 femur primordia correlates with lack of trichome formation in different Drosophila species, implying that Ubx inhibits formation of these structures. This role for Ubx, which has been termed a 'micromanaging role' is analogous to the function described here for Scr and Ubx in directing formation of T-rows in specific domains of T1 and T3 legs. Hence, micromanaging functions for Hox genes in generating complex and detailed morphologies may be a general phenomenon (Shroff, 2007).

Another common theme that has emerged from studies of the mechanisms through which Hox genes generate morphological diversity is that, in many cases, Hox genes function to suppress specific developmental pathways. For example, in legs, Antennapedia functions to repress expression of genes that promote antennal development, and Ubx functions to prevent development of specific macrochaete bristles on T3 legs. Furthermore, Ubx is known to act at several levels of the wing patterning hierarchy to suppress wing development in the haltere disc and as mentioned, Ubx functions late in leg development to suppress trichome formation. Less well understood, in contrast, is how and whether Hox genes act positively to direct the formation of morphological novelties among homologous structures, e.g., the T-row bristles on T1 and T3 legs. Further analysis of the mechanisms involved in T-row specification and morphogenesis is likely to provide insight into this question (Shroff, 2007).

Regulation of Delta during myogenesis

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).

Although the net effect of Ras and N signaling in the present system is the result of their antagonistic relationship, several forms of cooperative cross-talk also occur. For example, Ras activation induces the expression of Dl. Since the Ras signal is amplified by a positive feedback loop, this has the effect of biasing Dl expression to the emerging progenitor, thereby generating a polarized, nonautonomous inhibitory signal that acts on adjacent cells of the cluster. Aos is also a target of Ras activation, and Aos acts synergistically with the neurogenic pathway to block inductive RTK signaling. Thus, through its effects on the two inhibitory ligands, Dl and Aos, Ras cooperates with N to ensure that only one cell segregates as a progenitor from each equivalence group (Carmena, 2002).

Further cooperation is evident in the N-mediated down-regulation of Dl and Aos in prospective nonprogenitors, a combination of negative feedback and cross-talk that effectively prevents neighboring cells from sending an inhibitory signal to the emerging progenitor. Yan is yet another Ras-dependent component that reinforces the effect of N: when MAPK is suppressed in cells in which N is active, Yan is a functional repressor that blocks progenitor fate. Other examples of cooperation between Ras and N signaling include mammalian cell tumorigenesis and photoreceptor specification in the Drosophila eye (Carmena, 2002).

One seemingly paradoxical signaling interaction is N expression upregulation by Ras. Since Ras output is amplified in the progenitor, N protein might be expected to decrease in this cell, thereby restricting lateral inhibition to the appropriate direction. However, increased N in the Eve progenitor does not actually affect the polarity of lateral inhibition because the activating ligand, Dl, is downregulated by N in the adjacent nonprogenitors. Of further relevance, Dl may inhibit N activity when the two proteins are expressed in the same cell. Moreover, upregulation of N normally occurs very late in progenitor specification, as opposed to Dl which increases in one cell of the cluster at an earlier stage. Lastly, increased N has independent biological significance in progenitors since N is required for an asymmetric division that immediately follows the specification of these cells. In this respect, the response of the N receptor to Ras activation is an efficient, anticipatory 'feed forward' mechanism for insuring that this cell division is appropriately regulated (Carmena, 2002).

MicroRNA1 influences cardiac differentiation in Drosophila and regulates Notch signaling via post-transcriptional regulation of Delta

MicroRNAs (miRNAs) are genomically encoded small RNAs that hybridize with messenger RNAs, resulting in degradation or translational inhibition of targeted transcripts. The potential for miRNAs to regulate cell-lineage determination or differentiation from pluripotent progenitor or stem cells is unknown. MicroRNA1 (miR-1) is an ancient muscle-specific gene conserved in sequence and expression in Drosophila. Drosophila miR-1 (dmiR-1) is regulated through a serum response factor-like binding site in cardiac progenitor cells. Loss- and gain-of-function studies demonstrated a role for dmiR-1 in modulating cardiogenesis and in maintenance of muscle-gene expression. In vivo evidence is provided that dmiR-1 targets transcripts encoding the Notch ligand Delta, providing a potential mechanism for the expansion of cardiac and muscle progenitor cells and failure of progenitor cell differentiation in some dmiR-1 mutants. These findings demonstrate that dmiR-1 may 'fine-tune' critical steps involved in differentiation of cardiac and somatic muscle progenitors and targets a pathway required for progenitor cell specification and asymmetric cell division (Kwon, 2005).

The single orthologue of miR-1 in Drosophila, dmiR-1, is nearly identical in sequence to mouse and human miR-1. In situ hybridization revealed dmiR-1 transcripts in presumptive mesodermal cells as early as stage 5 (2.2-2.8 h) of Drosophila development. This pattern changed dynamically throughout gastrulation, but dmiR-1 consistently marked mesodermal cells. Transcripts persisted in later stages of cardiac and somatic (body wall) muscle differentiation, as in mice, and were also found in visceral muscles of the gut. dmiR-1 expression overlaps, but precedes, that of dmef2, a transcriptional regulator of muscle precursors (Kwon, 2005).

To determine whether transcriptional regulation of miR-1 is evolutionarily conserved, ten kb of genomic DNA surrounding Drosophila melanogaster and Drosophila pseudoobscura miR-1 genes were aligned to find regions of sequence conservation. Transgenic flies containing conserved islands 4.6 kb upstream of dmiR-1 adjacent to the gene encoding nuclear GFP (nGFP) recapitulates the endogenous dmiR-1 expression in all muscle types. Cardiac nGFP expression coincided with dmef2 expression in cardial cells and was present in pericardial cells, which do not express dmef2. The basic helix–loop–helix transcription factor Twist is essential for mesoderm specification and regulates dmiR-1 in certain domains, so the expression of nGFP driven by the dmiR-1 enhancer was directly compared with Twist expression. Whereas there was considerable overlap, the dmiR-1 enhancer directs expression in many areas of low Twist expression, including cardiac and visceral muscle progenitors, suggesting twist-independent regulation in these domains (Kwon, 2005).

Deletion analyses indicated that a 2.5-kb region was sufficient for expression in all domains of dmiR-1 expression except pericardial cells. Within this domain, a 720-bp genomic region containing a highly conserved SRF-like binding site recapitulates the expression directed by the 2.5-kb fragment. SRF, which is closely related to MEF2, controls the expression of genes involved in muscle differentiation, cell migration and proliferation. Prior studies showed that SRF is an obligate activator of miR-1 expression during cardiac development in the mouse. Mutation of the SRF-like site in flies abolishes nuclear GFP expression in cardiac and visceral muscle cells but not somatic muscle. In vitro, Drosophila SRF (DSRF) weakly activates transcription of a luciferase reporter through the SRF-like binding site. Addition of the potent cardiac and smooth muscle-specific SRF cofactor myocardin-related transcription factor robustly activated luciferase activity dependent on an intact SRF-like binding site. This observation is consistent with regulation of miR-1 in mice, but the possibility cannot be ruled out that Dmef2 also regulates cardial expression of dmiR-1 through this site independently or cooperatively with SRF (Kwon, 2005).

To begin to define the functions of dmiR-1 in vivo, two Exelixis lines of Drosophila containing FRT sites surrounding the dmiR-1 gene were used to generate a FRT-FLP-based deletion of the dmiR-1 locus. Successful excision of dmiR-1, the only known or predicted gene in the 31-kb deleted interval, was confirmed by sequence analysis and RT-PCR. Homozygous dmiR-1 deletion was 100% lethal, but a spectrum of severity was observed, with approximately one-third dying at embryonic stages, one-third around hatching, and the remaining at larval stages. Homozygous mutant larvae were abnormally lethargic compared with their heterozygous siblings before death. The embryonic and larval lethality was fully rescued by overexpression of dmiR-1 by using a mesoderm specific twi-Gal4 driver or by a 5.1-kb transgene encompassing the dmiR-1 genomic locus including the 4.6-kb enhancer and the sequence encoding dmiR-1, consistent with dmiR-1 being the sole gene within the deleted region responsible for the lethal phenotype. The variability in phenotype may be related to previously described maternal dmiR-1 transcripts, redundancy with other miRNAs or may simply reflect the role of dmiR-1 in 'fine-tuning' whether cells achieve the thresholds of critical proteins to initiate critical developmental events (Kwon, 2005).

Because one-third of all dmiR-1 mutants died around the time of hatching and another one-third at larval stages with poor mobility, whether there might be a discernable muscle defect was investigated. Nearly half of all dmiR-1 mutant embryos displayed severe defects in muscle gene expression with down-regulation of sarcomeric genes such as myosin heavy chain (MHC), indicating a late requirement for miR-1 to maintain muscle differentiation. This phenotype was also uniformly rescued by dmiR-1 under the control of the twist driver, indicating that the muscle differentiation defect was due to loss of dmiR-1 and not other sequences within the deleted region (Kwon, 2005).

Because miRNAs typically target numerous mRNAs, the phenotype of dmiR-1 mutants is likely due to down-regulation of multiple critical proteins. Despite the likely complexity of targets, attempts were made to identify mRNA targets of miR-1 in flies that might be involved in dmiR-1-dependent lineage determination and differentiation decisions. Although mouse miR-1 targets transcripts encoding the cardiac-enriched basic helix-loop-helix transcription factor Hand2, no miR-1-binding sites were identified in the 3'-UTR of Drosophila hand, suggesting alternative targets in flies. Because the more severe dmiR-1 gain- and loss-of-function phenotypes were reminiscent of progenitor defects induced by altering Notch signaling, the 3'-UTRs of genes involved in the Notch pathway were examined for potential sequence matching and accessibility to dmiR-1 (Kwon, 2005).

Several conserved putative miR-1-binding sites were found in the 3'-UTR of the gene encoding Delta, a membrane-bound ligand for Notch. Upon interaction with Delta, Notch is cleaved, allowing the Notch intracellular domain to translocate into the nucleus and regulate gene expression. Signaling between neighboring Delta- and Notch-expressing cells is necessary for lateral inhibition and asymmetric cell fates during lineage determination and involves repression of Delta in Notch-expressing cells and similar repression of Notch in adjacent Delta-expressing cells. Notch signaling also later regulates differentiation of numerous cell types, including cardial cells (Kwon, 2005).

Introduction of one of the putative dmiR-1-binding sites from the Delta 3'-UTR into the 3'-UTR of luciferase resulted in dose-dependent and specific down-regulation of luciferase activity in the presence of dmiR-1 in cultured fly S2 cells. Although the in vitro data supported Delta as a dmiR-1 target, attempts were made to determine whether dmiR-1 affected Delta protein levels in vivo. Available Delta antibodies are not sensitive enough to distinguish levels of Delta protein in embryonic muscle precursors. Therefore, an in vivo assay was used involving the well described role of Delta-Notch signaling in the developing wing disc, where disruption of Delta results in thickening of fly wing veins. Delta protein is normally detectable and expressed in two perpendicular stripes in the wing pouch. dmiR-1 was overexpressed along one of the two stripes using a dpp-Gal4 driver and the effects on Delta protein expression were assayed. Delta protein was markedly reduced exclusively in the domain of dmiR-1 expression, providing in vivo support for Delta as a target of miR-1 in flies. dmiR-1-induced loss of Delta in this specific subdomain of the wing resulted in thickening of wing veins, recapitulating the loss-of-Delta phenotype. The shortened-leg phenotype upon dmiR-1 overexpression provided further evidence of dmiR-1's effects on the Notch pathway, because this, too, was similar to the phenotype of flies lacking Delta. Together, the in vivo experiments provided compelling evidence that dmiR-1 can regulate Delta protein levels, providing a potential means to fine-tune cellular responses to Notch signaling. Given the recognized role of Notch signaling in asymmetric cell division of muscle progenitors, dmiR-1 regulation of Delta, along with other dmiR-1 targets, may be important in cardiac lineage determination events (Kwon, 2005).

Evidence that nervy, the Drosophila homolog of ETO/MTG8, promotes mechanosensory organ development by enhancing Notch signaling: nvy promote the SOP fate by transiently strengthening the Dl signal emanating from the SOP

In the imaginal tissue of developing fruit flies, achaete (ac) and scute (sc) expression defines a group of neurally-competent cells called the proneural cluster (PNC). From the PNC, a single cell, the sensory organ precursor (SOP), is selected as the adult mechanosensory organ precursor. The SOP expresses high levels of ac and sc and sends a strong Delta (Dl) signal, which activates the Notch (N) receptor in neighboring cells, preventing them from also adopting a neural fate. Previous work has determined how ac and sc expression in the PNC and SOP is regulated, but less is known about SOP-specific factors that promote SOP fate. This study describes the role of nervy (nvy), the Drosophila homolog of the mammalian proto-oncogene ETO, in mechanosensory organ formation. Nvy is specifically expressed in the SOP, where it interacts with the Ac and Sc DNA binding partner Daughterless (Da) and affects the expression of Ac and Sc targets. nvy loss- and gain-of-function experiments suggest that nvy reinforces, but is not absolutely required for, the SOP fate. A model is proposed in which nvy acts downstream of ac and sc to promote the SOP fate by transiently strengthening the Dl signal emanating from the SOP (Wildonger, 2005).

These results suggest that Nvy plays a role, albeit subtle, in the SOP's ability to send a strong Dl signal to neighboring cells. Although the data demonstrate that nvy is not required for the SOP fate, it is suggested that the ability of Nervy to increase the Dl signal sent by the SOP helps to reinforce the SOP fate. When nvy is ectopically expressed it completely inhibits the formation of mechanosensory organs. Using reagents that mark the PNC and SOP, it was found that ectopic Nvy blocks the formation of the SOP, but not the PNC. In contrast, elevating Nvy levels specifically within the SOP (using neur-Gal4) does not affect sensory organ development, indicating that ectopic Nvy blocks the formation of the SOP but does not inhibit its development once it is specified. Furthermore, ectopic Nvy does not block mechanosensory organ formation when Sens is also over-expressed, suggesting that ectopic Nvy blocks SOP formation before there are high levels of Sens in the nascent SOP. Consistent with this idea, no Sens expression is observed in the pnr domain of pnr-Gal4 UAS-nvy wing discs or in clones that ectopically express Nvy. These data suggest that ectopic Nvy interferes with SOP formation at a stage before Sens is expressed, which corresponds to when the SOP is initially specified (Wildonger, 2005).

nvy is normally expressed in the SOP shortly after Ac and Sc levels increase. Given the expression of endogenous nvy within the SOP, the following two possibilities werre considered to explain the ectopic Nvy phenotype and to gain some clues about wild type function of nvy. (1) It is possible that ectopic Nvy blocks SOP formation cell autonomously by inhibiting the expression of ac, sc, or their downstream targets (such as sens) that are necessary for SOP formation. (2) It is possible that ectopic Nvy acts cell non-autonomously by enhancing Dl signaling, resulting in the 'mutual inhibition' of cells expressing precociously high levels of nvy. A closer examination of clones that ectopically express Nvy revealed that SOPs were significantly less likely to form near the borders of Nvy expressing clones than control clones. These results suggest that Nvy is acting, at least in part, cell non-autonomously, perhaps by increasing the strength of the Dl signal (the possibility that Nvy may also act cell autonomously is discussed in the following section). As a test of this idea, Nvy was ectopically expressed in clones lacking nic, which encodes a transmembrane protein required for cleaving and activating N in response to ligand binding. Ectopic Nvy was unable to block SOP formation in nic mutant clones, demonstrating that Nvy's ability to block SOP formation requires the N signaling pathway to be intact. This finding is therefore consistent with the idea that Nvy normally enhances the level of active Dl in the SOP. Importantly, loss-of-function nvy experiments are also consistent with this proposed role for Nvy. Using two different methods to remove nvy (expressing nvy RNAi or generating clones of a nvy deficiency), it was found that PNC cells that neighbor nvy clones are more likely to adopt the SOP fate than PNC cells that neighbor wild type clones. This result is similar to what was observed when the relative amount of Dl differs between neighboring PNC cells: PNC cells that neighbor cells with less Dl are more likely to differentiate as SOPs. In contrast to the Dl experiments, however, the complete absence of nvy did not cause all PNCs to become SOPs. Keeping in mind that nvy expression is restricted to the SOP (nvy is not detectably expressed in the PNC), these data suggest that nvy is not a general regulator of Dl signaling throughout the PNC, but that nvy enhances Dl activity in the SOP when it is forming (Wildonger, 2005).

Although these experiments are consistent with the idea that nvy enhances Dl signaling in the SOP, no changes in Dl protein levels were directly detected in either nvy loss- or gain-of-function situations. There are several possible explanations for this negative result: (1) it is possible that nvy does affect Dl expression levels, but that the change is too slight or brief to distinguish with the available anti-Dl antibody; (2) nvy might not affect Dl expression, but affect its localization and/or signaling ability in a manner that cannot be detected in these experiments; (3) it is also possible that nvy does not affect Dl at all, but interacts with other factors to produce the phenotypes observed. It is suggested that experiments using VP16-Nvy help to distinguish between these possibilities. Expressing VP16-Nvy produces results opposite to those resulting from expressing Nvy: VP16-Nvy enhances E-lacZ expression, which ectopic Nvy represses, and its expression results in ectopic Sens+ SOPs. Based on these data and the evidence that ETO, the mammalian homolog of Nvy, acts as a transcriptional repressor, it is suggested that VP16-Nvy acts as a transcriptional activator of targets that wild type Nvy normally represses. When expressed in a PNC, VP16-Nvy strongly reduces the amount of Dl observed at the cell surface and in intracellular vesicles. This result suggests that wild type Nvy has the potential to affect Dl, although the result does not distinguish an effect on expression from an effect on protein stability or trafficking. That ectopic Nvy does not inhibit the expression of Dl-lacZ suggests that Nvy may be more likely to transcriptionally regulate a factor is involved in Dl stability or trafficking. Regardless of the mechanism, the finding that VP16-Nvy reduces Dl levels suggests that wild type Nvy has the potential to increase Dl levels, a proposal that is consistent with loss- and gain-of-function experiments (Wildonger, 2005).

The VP16-Nvy results, while consistent with the idea that Nvy affects Dl, do not explain why no change in Dl levels were detected in nvy loss- and gain-of-function experiments. Thus, it is thought that Nvy causes a small and/or transient increase in Dl activity (by affecting its expression, stability or localization). Nevertheless, no change in the amount or localization of Dl in wild type SOPs has been observed, despite genetic evidence that Dl signaling is a critical step in SOP fate determination. The lack of an observable change in Dl during wild type development, in combination with the current findings, lead to a proposal that the presumptive SOP may send a transient pulse of increased Dl signal that is sufficient to bias cell fates within the PNC. Nvy may, therefore, contribute to this transient pulse of Dl (Wildonger, 2005).

The experiments described here shed some light on the molecular activities Nvy has in the SOP. (1) Based on its ability to repress well-defined lacZ reporter genes, Nvy appears to be a transcriptional repressor, as is its mammalian homolog ETO. (2) This study shows that ectopic Nvy appears to interfere with the function (as opposed to the expression) of Ac and Sc because re-supplying Ac and Sc in pnr-Gal4 UAS-nvy flies was unable to rescue the bald phenotype. In contrast, expression of Da, a bHLH DNA binding partner for Ac and Sc, was able to partially rescue the bald phenotype of pnr-Gal4 UAS-nvy flies. Moreover, nvy and da were found to genetically interact (e.g., reducing nvy levels enhanced a da gain-of-function phenotype), and Nvy and Da were found to physically interact. These findings are consistent with a recent report showing that ETO directly interacts with HEB, a bHLH factor in the same class as Da. The domain through which ETO interacts with HEB (and other mammalian class I bHLH transcription factors) is conserved in Nvy, and HEB's ETO interaction domain is found in Da. These data lead to a proposal that Nvy, a presumptive transcriptional repressor, has the ability to function with Ac/Da and Sc/Da heterodimers to repress the transcription of some target genes. In the absence of Nvy, such as in the non-SOP cells of a PNC, Ac/Da and Sc/Da may have the potential to activate these same target genes. However, these experiments also suggest that the interaction between Nvy and Da may not be required for all of Nvy's functions because VP16-Nvy is able to lower Dl levels even in da mutant clones. One potential explanation for this Da-independent function is that Nvy may be able to directly interact with DNA. In summary, it is speculated that the Nvy–Da interaction is only required for the regulation of a subset of target genes (Wildonger, 2005).

The proposal that Nvy works with Ac/Da and Sc/Da to repress target genes may on the surface seem at odds with the suggestion that Nvy can transiently increase the levels of Dl, because it is thought that Ac/Da and Sc/Da heterodimers activate Dl expression in the SOP. However, it is not known if Dl levels are in fact directly increased by Ac/Sc. It is stressed that the timing of expression of these genes is critical to understanding how they function in vivo. Based on the wild type timing of its expression, nvy is likely to be a target of Ac/Sc in the presumptive SOP. Accordingly, there will be a window of time when Ac/Sc levels are high and Nvy levels are low in the presumptive SOP. This window of time may be sufficient for Ac/Sc to affect Dl expression and initiate the bias in favor of the SOP fate. Once Nvy levels increase, it may then work with Ac/Sc to repress the expression of some target genes, some of which may cause a further increase in Dl signaling. However, it is hypothesized that nvy's role in this process is after the bias has already been initiated (Wildonger, 2005).

In summary, it is suggested that Nvy plays a subtle but observable role in the establishment of the SOP fate. Although it is not essential for the SOP fate, it may be that Nvy helps the SOP/non-SOP bias by increasing the strength of the Dl signal sent by the SOP. Because nvy is evolutionarily conserved, both in its protein sequence and nervous system expression, it is suggested that this role, although subtle, is important for the stereotyped uniformity of mechanosensory organ development. In addition, nvy may also play a role in later stages of neurogenesis, in particular axon pathfinding. Because of Nvy's role as a transcriptional repressor, it is further suggested that Nvy increases the Dl signal indirectly, by repressing a gene (factor X) that normally inhibits Dl activity. Based on Nvy's ability to interact with Da, this hypothetical target may be repressed by Nvy in combination with Ac/Da and Sc/Da heterodimers. Interestingly, it follows that in non-SOP cells of the PNC, which express ac and sc but not nvy, this hypothetical target may continue to be expressed, helping to downregulate Dl activity in these cells and thereby further increase the SOP/non-SOP bias. Clearly, the test of this proposal requires the identification of factor X as well as a more detailed understanding of how Dl levels and activity are modulated in the SOP (Wildonger, 2005).

Hindsight modulates Delta expression during Drosophila cone cell induction

The induction of cone cells in the Drosophila larval eye disc by the determined R1/R6 photoreceptor precursor cells requires integration of the Delta-Notch and EGF receptor signaling pathways with the activity of the Lozenge transcription factor. This study demonstrates that the zinc-finger transcription factor Hindsight (Hnt) is required for normal cone-cell induction. R-cells in which hindsight levels are knocked down using RNAi show normal subtype specification, but these cells have lower levels of the Notch ligand Delta. HNT functions in the determined R1/R6 precursor cells to allow Delta transcription to reach high enough levels at the right time to induce the cone-cell determinants Prospero and D-Pax2 in neighboring cells. The Delta signal emanating from the R1/R6 precursor cells is also required to specify the R7 precursor cell by repressing seven-up. As hindsight mutants have normal R7 cell-fate determination, it is inferred that there is a lower threshold of Delta required for R7 specification than for cone-cell induction (Pickup, 2009).

This study shows that Hnt function is necessary to elevate the Dl ligand in the R1/R6 precursor cells to a level high enough to achieve cone-cell induction. Notably, Hnt is not an on/off switch for Dl expression; rather it potentiates the level of Dl transcription in the R1/R6 precursor cells. The data suggest that this modulation is likely to be independent of Chn, which is itself a transcriptional repressor of Dl. Although this paper does not show that this Hnt effect is due to direct action, the exact sequence for two Hnt binding sites was found in the upstream and intronic sequences of the Delta transcription unit (Pickup, 2009).

Earlier reports describing Hnt function in the ovary show that Hnt expression is regulated by the Notch signaling pathway and controls follicle cell proliferation and differentiation. This paper reports that Hnt acts upstream of Notch activation by regulating Dl ligand expression levels. These two modes of regulation are not necessarily mutually exclusive, but it is not thought that Notch activates the hnt gene in the eye. (1) Hnt is expressed in all the R-cell precursors in the eye, whereas the Notch pathway is activated at high levels only in a subset of these precursors, as well as in the accessory cone and pigment cell precursors, where Hnt is not expressed at all. (2) When Notch activity is attenuated by using the Nts mutant, Hnt expression in the furrow expands to all cells that now acquire a neuronal fate. This result cannot be interpreted as a simple repression of Hnt expression by Notch activation in non-neuronal cells, as Hnt expression is not complementary to Notch activation in the eye disc. (3) Notch activation cannot be sufficient to induce Hnt expression in the eye disc, since no expansion of Hnt expression into adjacent, non-determined cells is seen when Dl is ectopically expressed early in the cone-cell precursors (with the lz-Gal4 driver). (4) It was shown that the expression of Dl in the R-cell precursors is partly dependent on Hnt function. Others have clearly demonstrated that this late Dl expression does not require Notch activity, since it is unaffected in a Nts1 mutant (Pickup, 2009).

The two-signal model of R7 fate hypothesizes that R7 determination requires a strong RTK signal (achieved by the additive effects of Sevenless and EGFR activation) together with Notch activation. These signals are necessary to activate pros and repress svp expression, respectively. Since the cone-cell precursor cells do not contact the determined R8 cell at the appropriate time, they will not 'see' the SEV ligand BOSS. Cone cell precursors, then, will not ordinarily activate their Sev receptors. In this model, different fates have been reinforced in the R7/cone equivalence group by adding a second, activating ligand for EGFR (Pickup, 2009).

This paper suggests a further level of complexity. It was shown, by manipulating the level of Dl in the R1/R6 signaling cells, that activation of the key players in cone-cell determination requires high levels of the Notch activation in the cone-cell precursor cell. Several lines of evidence support the idea that the level of the Dl ligand is translated into cell-fate differences in a responding R precursor cell. Since there is low Dl expression in the R7 precursor cell and only late expression of Dl in the cone-cell precursor cell, the adjacent R1/R6 precursor cells never activate their Notch receptors. Both the R7 precursor and the cone-cell precursor cells receive their ligand signal from the R1/R6 precursor cells. In this hypothesis, the R7 precursor cell requires only a low level of ligand signal to activate the R7-like program: turning on pros and off svp (Pickup, 2009).

It is suggested that the cone-cell precursor requires a high level of ligand signal to activate the cone-cell program. Expressing a dominant-negative form of Dl in the R1/R6 signaling cells prevents cone-cell, but not R7-cell, determination. Since both the cone and R7 precursor cells receive their Dl input from the same R1/R6 cells, it is possible that an intrinsic feature of the R7 precursor cell - possibly the high RTK activation - antagonizes N signaling, so that D-Pax2 transcription does not occur in that cell. The transcriptional repressor, Lola, may also be involved in this distinction, since it is known to bias precursor cells towards R7-over cone-cell fate (Pickup, 2009).

Although a role for Notch signaling in cone-cell induction has been shown to be necessary for D-Pax2 expression, it has not been directly demonstrated as necessary for pros regulation in cone cells. The experiments presented in this study suggest that high levels of Notch signaling may indirectly or directly be required for Pros expression in the cone-precursor cells. This requirement is independent of the role of SU(H) in inducing D-Pax2, since there are normal levels of Pros in the cone-cell precursors of a D-Pax2 null mutant. Ectopically activating the Notch pathway in the R1/R6 precursor cells occasionally induces ectopic Pros (but eliminates ELAV) in these cells. Although this effect on Pros expression may be a secondary result of a cell-fate transformation, it could also be interpreted as a more direct effect of Notch signaling on pros transcription. In a different context, Pros expression has been shown to be affected by Dl-activated Notch signaling in a subset of glial cells in the embryonic CNS (Pickup, 2009).

Why would there be two Dl thresholds for different cell fates? There is some preliminary work that suggests different mechanisms for Notch-activated transcriptional readout in the responding cell, depending on the level of signal received. In the cone-cell equivalence group, the cone-cell determination pathway requires that D-PAX2 and Pros be expressed. It is hypothesized that D-Pax2 may require a higher level of Notch activation than Pros, which is also required for R7 determination. These experiments indicate that there may be coordinated regulation of both D-Pax2 and Pros expression in the cone cells. It is postulated that the mechanism of Pros-gene induction in the cone cells is different from pros regulation in R7. By potentiating the level of Dl gene expression in the R1/R6 signaling cells, it is possible to overlay the cone-cell fate over the transcriptional module necessary for R7-cell fate. This simple change has, thus, allowed for the elaboration of very different cell fates from the same equivalence group (Pickup, 2009).

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

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

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

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

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

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

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

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

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

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

odd-skipped genes and lines organize the notum anterior-posterior axis using autonomous and non-autonomous mechanisms>

The growth and patterning of Drosophila wing and notum primordia depend on their subdivision into progressively smaller domains by secreted signals that emanate from localized sources termed organizers. While the mechanisms that organize the wing primordium have been studied extensively, those that organize the notum are incompletely understood. The genes odd-skipped (odd), drumstick (drm), sob, and bowl comprise the odd-skipped family of C2H2 zinc finger genes, which has been implicated in notum growth and patterning. This study shows that drm, Bowl, and eyegone (eyg), a gene required for notum patterning, accumulate in nested domains in the anterior notum. Ectopic drm organized the nested expression of these anterior notum genes and downregulated the expression of posterior notum genes. The cell-autonomous induction of Bowl and Eyg required bowl, while the non-autonomous effects were independent of bowl. The homeodomain protein Bar is expressed along the anterior border of the notum adjacent to cells expressing the Notch (N) ligand Delta (Dl). bowl was required to promote Bar and repress Dl expression to pattern the anterior notum in a cell-autonomous manner, while lines acted antagonistically to bowl posterior to the Bowl domain. These data suggest that the odd-skipped genes act at the anterior notum border to organize the notum anterior–posterior (AP) axis using both autonomous and non-autonomous mechanisms (Del Signore, 2012).

In many developmental processes, signals that emanate from field borders play a crucial instructive role in patterning morphogenetic fields. The early Drosophila embryo is patterned by opposing gradients of Bicoid and Nanos that are generated from localized translation of corresponding mRNAs at the anterior and posterior poles of the embryo. In the embryonic epidermis, the pattern of cell differentiation across each segment is regulated by the secreted Wg and Hh signals that emanate from localized sources at the anterior and posterior borders of each segment. Similarly, the dorsoventral axis of the vertebrate spinal cord is organized by Shh ventrally, and BMP and Wnt signals that emanate from localized dorsal sources. By contrast, current models of notum AP patterning focus mainly on the organizing influence of Dpp, which is secreted from the posterior border of the notum. Previous work has found that odd-skipped genes are expressed along the anterior border of the notum, and that broadly inhibiting their function in early wing discs caused a severe reduction or complete loss of the notum. As this reduction occurred despite the maintenance of dpp expression (Nusinow, 2008), whether the odd-skipped genes might define a second organizing center within the developing notum was investigated. The current findings indeed suggest that signals that emanate from the anterior border of the notum act reciprocally to Dpp to promote expression of anterior notum genes and repress expression of posterior genes. Through loss- and gain-of-function clonal analyses, it was demonstrated that the odd-skipped genes pattern the notum AP axis both locally through regulation of Eyg, Bar, and Dl, and broadly through the regulation of Eyg and Tup. Finally, it was shown that lines acts antagonistically to bowl in this process (see Model of the role odd-skipped genes in notum AP patterning) (Del Signore, 2012).

drm overexpression was sufficient to promote Eyg accumulation non-autonomously within the notum. This activity suggests that drm controls expression of an unidentified signal that spreads from the drm domain to induce Eyg accumulation non-autonomously. Alternatively, drm could initiate the propagation of a cascade of local inductive interactions to induce Eyg at a distance. Recent studies have shown that recruitment of cells to the wing field is accomplished by the propagation of a feed forward signal from the DV compartment boundary. In this process signaling at the border between Vestigial (Vg) and non-Vg expressing cells is used to recruit non-Vg expressing cells to the expanding wing field, a process dependent on signaling through the Fat-Dachsous pathway. Though a functional relationship between odd-skipped genes and Ft-Ds signaling has yet to be characterized, it is interesting to note that Ds accumulates in a complex graded AP pattern across the notum, consistent with such a role (Del Signore, 2012).

In addition to the broad induction of Eyg accumulation, it was surprising to find that drm overexpression also induced Bowl in cells just adjacent to clones. Though the effect was subtle, it is noted that this pattern of activation recapitulated the endogenous nested pattern of drm and Bowl expression in the presumptive prescutum. It is unclear whether the nested expression of odd-skipped genes plays a functional role in notum AP patterning. Despite this, the concordance of endogenous and ectopic expression patterns supports the hypothesis that ectopic drm induces a physiologically relevant program of anterior gene expression in the notum. One possible clue as to the relevance of this nested pattern may come from the observation that only drm was able to promote Bowl non-autonomously. In contrast, lines−/−, odd+, and sob+ clones each induced only cell-autonomous accumulation of Bowl. Notably, these clones rounded up and segregated from the epithelium, while drm expressing clones remained integrated with the surrounding epithelium. One interpretation of these data is that abrupt discontinuities in the level of Odd-skipped proteins may alter epithelial morphology. This interpretation is further supported by the observation that bowl mutant clones within the Bowl domain adopt a compact, round morphology relative to clones outside the Bowl domain. It is hypothesized that drm promotes lower levels of Bowl in nearby cells to dampen otherwise sharp discontinuities in Bowl activity to regulate local buckling of the epithelium (Del Signore, 2012).

Alternatively, differences in the total levels or ratios of Odd family proteins along the anterior border of the notum could elicit different transcriptional outcomes. Since Odd and Bowl have been shown to interact with the transcriptional co-repressor Groucho, variation in the levels of the Odd-skipped proteins could titrate Groucho and affect Groucho-dependent transcriptional outputs. Alternatively, given their distinct structure outside the zinc finger domain, the Odd-skipped proteins could interact with distinct sets of target genes to pattern the anterior border of the notum. Though additional experiments will be required to ascertain whether such mechanisms are active in the prescutum, this study provides evidence that bowl is strictly required for the early autonomous induction of Eyg, the later expression of Bar genes, and the repression of Dl. These results provide evidence that odd-skipped genes act both independently and redundantly to organize the notum AP axis (Del Signore, 2012).

bowl is essential for patterning the prescutum, but not for broadly patterning the notum AP axis. Previous studies have revealed a variety of essential and redundant functions for odd-skipped family genes in patterning embryonic and larval tissues. In the embryo, drm and bowl antagonize lines function to pattern the dorsal embryonic epidermis, foregut, and hindgut, while odd functions as a pair rule gene to promote embryonic segmentation. In the leg imaginal disc, bowl is essential for patterning the tarsal proximodistal axis at early stages, but acts redundantly with other odd-skipped genes to control leg segmentation later in developmen. In the eye, bowl is essential for the initiation of retinogenesis from the eye margin, while odd and drm have been proposed to activate Bowl redundantly (Del Signore, 2012).

Loss-of-function analysis revealed that neither drm nor odd is necessary to stabilize Bowl. At present the possibility cannot be excluded that sob is necessary to promote Bowl accumulation because a null sob mutant is not yet available. Biochemical and genetic analysis demonstrates that not only Drm, but also Odd and Sob can each outcompete the interaction of Lines with Bowl and stabilize the Bowl proteins in S2 cells and in vivo. These results suggest that different combinations of Odd-skipped proteins could be used to activate bowl depending on context (Del Signore, 2012).

Previous work suggested reciprocal roles for lines and odd-skipped genes in subdividing the early wing disc into disc proper and peripodial epithelium. The loss-of-function analysis described in this study suggests that the odd-skipped genes act redundantly to control the early specification of the PE and the subsequent expansion of the notum, while revealing an essential role for bowl in specification of the anterior prescutum. Redundancy can increase the robustness of essential developmental processes and provide a buffer against fluctuations in activity of single genes. The redundant role of the odd-skipped genes in PE specification and notum expansion could therefore serve to ensure the optimal growth of the wing disc at early stages and that of the notum at later stages and protect these critical processes from perturbations (Del Signore, 2012).

It is concluded that the growth and patterning of the wing field are coordinated with the elaboration of the wing PD axis. The developing notum lacks an obvious PD axis, and instead is subdivided into a series of AP and mediolateral domains. The establishment of organizers that act antagonistically from opposing field borders is a robust strategy to subdivide the notum AP axis. This work demonstrates that the odd-skipped genes act autonomously at the anterior border of the notum to specify the prescutum, and non-autonomously at short and long range to control the expression of transcription factors that prefigure the differentiation of the notum AP axis. Though further experiments will be required to characterize the mechanism by which this putative organizer acts, these studies provide evidence that the anterior border of the notum exhibits the functional attributes of an organizer (Del Signore, 2012).

Targets of Activity

The role of the Notch and Wingless signaling pathways has been investigated in the maintenance of wing margin identity through the study of cut, a homeobox-containing transcription factor and a late-arising margin-specific marker. By late third instar, a tripartite domain of gene expression can be identified in the area of the dorsoventral compartment boundary, which marks the presumptive wing margin. A central domain of cut- and wingless-expressing cells is flanked on the dorsal and ventral side by domains of cells expressing elevated levels of the Notch ligands Delta and Serrate. Cut acts to maintain margin wingless expression, providing a potential explanation for the cut mutant phenotype. Notch, but not Wingless signaling, is autonomously required for cut expression. Rather, Wingless is required indirectly for cut expression; the results suggest this requirement is due to the regulation by wingless of Delta and Serrate expression in cells flanking the cut and wingless expression domains. Delta and Serrate play a dual role in the regulation of cut and wingless expression. Normal, high levels of Delta and Serrate can trigger cut and wingless expression in adjacent cells lacking Delta and Serrate. However, high levels of Delta and Serrate also act in a dominant negative fashion, since cells expressing such levels cannot themselves express cut or wingless. It is proposed that the boundary of Notch ligand along the normal margin plays a similar role as part of a dynamic feedback loop that maintains the tripartite pattern of margin gene expression (Micchelli, 1997).

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

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

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

Thus a single row of boundary cells forms in between En-expressing dorsal cells and Delta-expressing ventral cells and is determined by Notch signaling. Unlike in wing imaginal disc development, Ser seems not involved, and the glycosyltransferase Fringe plays only a minor role for the proper positioning of the DV boundary in the large intestine. These results rather suggest two major determinants that control where Notch signaling can occur in the hindgut: (1) the localization of Delta, which is expressed in ventral cells at high levels and not expressed (or at very low levels) in the adjacent boundary cells in which Notch signaling is eventually activated; (2) the Crb-dependent transport of the Notch receptor to the apical membrane domain of the boundary cells. How the initial En and Dl expression domains are set up in the large intestine is not known. It is noted, however, that the En expression domain in the hindgut primordium is initially broader in early stage 7 embryos and only subsequently refines to the dorsal cells, whereas Dl expression is confined to ventral cells from early stage 7 onward. It is thus possible that Notch signaling in the boundary cells leads to a cell-autonomous repression of en expression, consistent with the repression of en upon Nicd overexpression. Immunhistological studies show that the proper specification of boundary cells in crb mutants correlates with the apical localization of the Notch receptor. The finding that Crb expression in the boundary cells depends on Notch signaling suggests the possibility of a feedback loop that ensures proper receptor localization required for establishing the competence of the boundary cells to receive the Delta signal. It cannot be excluded, however, that the failure of Notch signaling in crb mutants may also be caused by the mislocalization of other localized proteins. It is noteworthy that the apical side of the boundary cells faces the lumen of the hindgut. Activating Notch receptors along the entire apical plate of the boundary cells would therefore require also a secreted form of Delta. The extracellular domain of Delta has been found as a soluble product in the supernatant of Drosophila cultured cells and in embryonic extracts, and has been shown to arise by a proteolytic activity of the ADAM metalloprotease Kuzbanian. Both soluble forms of Delta and Serrate are able to act as antagonists and agonists of the Notch pathway in vivo. It is possible that such a form of Delta and/or additional apically localized factors are involved in binding and activating the Notch receptor locally in the boundary cells of the hindgut (Fusse, 2002).

These results provide evidence that Notch signaling in the Drosophila hindgut controls the fate of a single row of boundary cells separating the dorsal and ventral halves of the gut tube. Activation of the Notch receptor in the boundary cells is mediated by its ligand Delta that is expressed in adjacent ventral cells. The induction of Notch target genes activate the expression of the apical polarity organizer Crb, which may be required, in turn, for apical Notch receptor localization. These findings identify a simple mechanism that controls the specification of a single row of DV boundary cells in an animal gut (Fusse, 2002).

The microRNA pathway regulates the temporal pattern of Notch signaling in Drosophila follicle cells

Multicellular development requires the correct spatial and temporal regulation of cell division and differentiation. These processes are frequently coordinated by the activities of various signaling pathways such as Notch signaling. From a screen for modifiers of Notch signaling in Drosophila the RNA helicase Belle, a recently described component of the RNA interference pathway (Ulvila, 2006; Zhou, 2008), was identified as an important regulator of the timing of Notch activity in follicle cells. Loss of Belle delays activation of Notch signaling, which results in delayed follicle cell differentiation and defects in the cell cycle. Because mutations in well-characterized microRNA components phenocopied the Notch defects observed in belle mutants, Belle might be functioning in the microRNA pathway in follicle cells. The effect of loss of microRNAs on Notch signaling occurs upstream of Notch cleavage, as expression of the constitutively active intracellular domain of Notch in microRNA-defective cells restored proper activation of Notch. Furthermore, evidence is presented that the Notch ligand Delta is an important target of microRNA regulation in follicle cells and regulates the timing of Notch activation through cis inhibition of Notch. This study has uncovered a complex regulatory process in which the microRNA pathway promotes Notch activation by repressing Delta-mediated inhibition of Notch in follicle cells (Poulton, 2011).

The strict regulation of important cellular processes, such as the temporal activity of signaling pathways like Notch, is an essential point of control in guiding the development of multicellular organisms. Cells have therefore evolved a complex array of mechanisms to regulate signaling pathways. miRNA regulation of gene expression has rapidly emerged as one of the most important of these regulatory mechanisms. This study has shown that the correct timing of Notch activity in follicle cells requires the miRNA pathway and the newly identified RNAi component Bel. The data suggest that one important target of miRNA-based regulation of Notch signaling in follicle cells is Delta, in which Delta acts as a repressor of Notch activity (Poulton, 2011).

These findings that two core components of miRNA production are required to properly initiate the mitotic-to-endocycle switch in follicle cells by promoting Notch signaling describe a novel mechanism by which the miRNA pathway regulates this key developmental event. Interestingly, the miRNA pathway appears to control the overall timing of Notch activity, as disruption of the miRNA pathway results in a delay of Notch activation and inactivation in follicle cells. Previous work has shown that certain miRNAs, known as heterochronic miRNAs, regulate the timing of important developmental processes on a wide biological scale, from changes in cell cycle to the transition from juvenile to adult. This research identifies a new example of heterochrony mediated by miRNAs, in which cell cycle switches and differentiation are shifted in time as a result of delayed Notch signaling activity (Poulton, 2011).

Bel is a DEAD-box RNA helicase that was recently identified in two Drosophila cell culture screens as necessary for effective siRNA knockdown (Ulvila, 2006; Zhou, 2008). Precisely how Bel functions in this process is unknown, although data from the Zhou screen suggest that Bel acts downstream of siRNA production and loading. Interestingly, although Bel did not significantly disrupt miRNA-based assays in that screen, Bel was found to be in a complex with components of both the miRNA and siRNA pathways, and Bel immunoprecipitation pulled down both miRNAs and siRNAs, suggesting that Bel might be involved in both pathways. The similarities described between the bel mutant phenotype and the phenotypes of the miRNA pathway components Dicer (Dcr-1) and pasha imply that Bel might function in the miRNA pathway. Attempts were made to test the role of Bel in the miRNA pathway more directly using the GFP-tagged Delta 3'UTR sensor line, the expression of which is regulated by miRNA activity, but the results of these experiments were inconclusive. Although Bel appears to function in the siRNA pathway, this study found that the siRNA pathway is not involved in regulating Notch in follicle cells. A few reports have also identified several phenotypes associated with disruption of bel that indicate that Bel functions in the G1/S transition in the eye disc by affecting Hedgehog signaling and Dacapo expression (Ambrus, 2010; Ambrus, 2007), as well as identifying a role for Bel with the zinc-finger protein Zn72D in regulating the splicing and translation of maleless transcripts (Worringer, 2009). It will be interesting to determine whether the function of Bel in these other important cellular processes is also related to a role in RNAi pathways (Poulton, 2011).

Notch can be both activated and inhibited by its ligands. In oogenesis, it is known that Delta from the germline cells functions in trans to activate Notch in the surrounding follicle cells. This study found that Delta expressed in the follicle cells operates in its repressive capacity to prevent premature activation of Notch. Because Delta is actually upregulated in the germline by stage 5/6, well before the expression of Notch target genes at stage 7, and in light of the data on the inhibitory role of follicle cell Delta, it is likely that the presence of Delta from the germline alone is not what determines the precise timing of Notch activity. Instead, a model is favored in which the timing of Notch activity is determined by a titration of trans-activating germline Delta relative to the cis-inhibitory effects of follicle cell Delta. Therefore, loss of follicle cell Delta, as in the Delta mutant clone experiments, allows earlier activation of Notch by the lower levels of Delta presented by the germline before stage 7, as well as higher levels of Notch activity relative to wild-type cells in mid-oogenesis. This antagonistic relationship between germline and follicle cell Delta suggests that there must be a precise balance between these two populations of Delta that determines exactly when Notch is activated during oogenesis; analysis of the miRNA pathway suggests that miRNAs might help to fine-tune this balance (Poulton, 2011).

The conclusion that Delta is a relevant target of miRNA-based control of Notch activity in follicle cells is supported by the following observations. First, expression of NICD is sufficient to restore proper activation of Notch in the Dcr-1 mutant, indicating that the relevant miRNA target functions upstream in the Notch pathway (prior to ligand-induced Notch cleavage). Because ligand-based inhibition by Delta acts upstream of Notch cleavage, Delta is a logical candidate of miRNA regulation. Second, Delta,Dcr-1 double-mutant analysis strongly suggests that Delta is an important target of miRNAs. Specifically, in Dcr-1 single-mutant clones, Notch signaling is delayed, yet removal of Delta along with loss of Dcr-1 leads to premature activation of Notch, as seen in Delta single-mutant clones. This indicates that the inhibitory effects on Notch signaling caused by loss of miRNAs requires the presence of Delta. However, the possibility cannot be ruled out that the activating effects of loss of Delta on Notch might be stronger than the inhibitory effects of loss of miRNAs on repressing Notch activity through some other miRNA target. Third, Delta is an apparent direct target of the miRNA pathway, as indicated by experiments demonstrating that follicle cell clones of Dcr-1 and pasha result in increased Delta protein and increased expression of a Delta 3'UTR sensor. Together, the ectopic expression of Delta protein and of the Delta 3'UTR sensor in the Dcr-1,pasha clones, in conjunction with the Delta,Dcr-1 double-mutant analysis, strongly suggest that the miRNA pathway regulates Notch activity by repressing Delta protein levels (Poulton, 2011).

Cis inhibition of Notch has also been described for Ser, raising the possibility that Ser might be functioning in follicle cells in a similar capacity to that which was discovered for Delta. However, Ser mutant follicle cell clones possess no defects in Notch activity markers. To determine whether Ser is repressed by the miRNA pathway in follicle cells, Ser protein levels were examined in follicle cells double mutant for Dcr-1 and pasha, and no changes were observed in Ser expression, which in the wild type was essentially undetectable. It is concluded that Ser does not play a role in regulating Notch activity in follicle cells (Poulton, 2011).

More than two dozen miRNAs are predicted to target Delta mRNA. Owing largely to a lack of readily available mutants to conduct a thorough loss-of-function screen for the miRNA(s) involved, it remains unknown which miRNAs are important in governing the timing of Notch signaling in follicle cells. Both loss of function and overexpression of miR-1, which has been previously demonstrated to regulate Delta in Drosophila heart development, were tested; however, neither produced any phenotype consistent with the described Notch defects. As the genetic tools available to investigate the roles of specific miRNAs improve, and the ability to predict which miRNAs target certain transcripts also improves, it should be possible to identify the relevant miRNAs involved in this process (Poulton, 2011).

These findings describe a complex system by which developing egg chambers regulate the timing of several key events, including cell cycle programs and differentiation. Mechanistically, it was found that the miRNA pathway controls the temporal pattern of Notch activity, apparently by limiting Delta protein levels in follicle cells, in which Delta exerts an inhibitory effect on Notch. The data support a model in which the timing of Notch activation is determined not just by the expression of germline Delta, but also by a multi-layered regulatory system in which follicle cell Delta prevents premature Notch activation, while miRNAs serve to counter this inhibitory effect by limiting Delta expression. Such a model of miRNA function in follicle cells fits well with the developing theme that miRNAs commonly serve to fine-tune developmental processes by subtle regulation of key regulators. It will be interesting to determine whether miRNAs also regulate Notch signaling in other tissues of the fly through a similar mechanism of ligand-mediated inhibition of Notch, and it will be particularly exciting to investigate whether this regulatory network is utilized in other animals (Poulton, 2011).

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

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