Delta
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).
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).
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: 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).
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).
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).
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).
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).
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).
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).
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 suggest
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).