RNA in situ hybridization was used to examine the spatiotemporal expression pattern of dCul-3 transcripts in Drosophila embryos and imaginal discs. dCul-3 mRNA is expressed ubiquitously throughout all stages of embryonic development and in imaginal discs. Notably, high levels of dCul-3 mRNA are observed in 0-1 h old embryos indicating significant maternal deposit of dCul-3 mRNA into oocytes. Consistent with RNA in situ hybridization results, dCul-3 protein is detected in the cytoplasm of all cells in embryos and imaginal discs (Mistry, 2004).
Sequence analysis of the two longest cDNAs has identified a short 80 nucleotide cryptic exon flanked by non-consensus splice sites. Retention of the putative exon and in-frame stop codons would truncate dCul-3 prior to the conserved C-terminal domain whereas utilisation of the non-consensus splice sites would produce full-length dCul-3. Although RT-PCR experiments suggested the possibility of multiple dCul-3 protein isoforms, Western analyses on total protein from wild-type (Oregon-R) embryos using antibodies specific to N- and C-terminus of dCul-3 identified a single 80.9 kDa band that appears to correspond to the full-length form of dCul-3. These results indicate that dCul-3 exists primarily, if not exclusively, as a single protein isoform in Drosophila (Mistry, 2004).
Differentiation of the Drosophila retina occurs as a morphogenetic furrow sweeps anteriorly across the eye imaginal disc, driven by Hedgehog secretion from photoreceptor precursors differentiating behind the furrow. A BTB protein, Roadkill, is expressed posterior to the furrow and targets the Hedgehog signal transduction component Cubitus interruptus for degradation by Cullin-3 and the proteosome. Clonal analysis and conditional mutant studies establish that roadkill transcription is activated by the EGF receptor and Ras pathway in most differentiating retinal cells, and by both EGF receptor/Ras and by Hedgehog signaling in cells that remain unspecified. These findings outline a circuit by which Hedgehog signal transduction is modified as Hedgehog signaling initiates retinal differentiation. A model is presented for regulation of the Cullin-3 and Cullin-1 pathways that modifies Hedgehog signaling as the morphogenetic furrow moves and the responses of retinal cells change (Baker, 2009).
As the morphogenetic furrow crosses the eye disc, Ci155 accumulates most highly just anterior to the morphogenetic furrow, even though Hh is secreted posterior to the morphogenetic furrow. The sharp reduction in Ci155 as the furrow passes is associated with a switch from Cul1-dependent processing to Cul3-dependent degradation (Ou, 2002). The posterior eye expresses rdx, encoding a BTB protein that couples Ci 155 to the Cul3 pathway (Kent, 2006; Zhang, 2006). This study identified the signals that induce rdx and that process Ci155 in the posterior eye (Baker, 2009).
The induction of rdx transcription couples Ci155 processing to Cul3 (Kent, 2006; Zhang, 2006). rdx transcription is regulated by both Hh signaling and Ras signaling, and there were distinctions between cell types. The smo mosaic and hhts2 experiments show that Hh signaling is continuously required for rdx transcription in unspecified cells with basal nuclei. In the absence of smo, EGFR-dependent rdx transcription occurs in differentiating photoreceptor cells only, not in unspecified cells. The egfr mosaics show that EGFR is essential for rdx transcription in all cells except the R8 photoreceptor class. Thus, EGFR-dependent differentiation was sufficient to induce rdx in photoreceptors even without Hh signaling, but Hh was not sufficient to induce rdx anywhere without EGFR signaling, except for the R8 cells. Undifferentiated cells might require both the Ras and Hh signaling pathways to induce rdx because the level of Ras signaling is lower in unspecified cells than in differentiating cells of the ommatidia. Alternatively, there may be a combinatorial requirement for both pathways in unspecified cells (Baker, 2009).
There has been some discussion of whether proteolysis of Ci155 by Cul-3 is regulated directly by Hh, as is Cul-1 dependent Ci processing. The current studies provide no support for this idea. In all the genotypes examined, Ci proteolysis correlates with the expression of rdx, and the simplest explanation is that the only effect of Hh on the Cul-3 pathway is through rdx transcription, directly in unspecified cells, and indirectly via EGFR-mediated differentiation in most specified cells (Baker, 2009).
Two mechanisms, acting in different cells, appear to reduce Hh responses through Ci155 after the furrow passes. One also occurs in wing development, where rdx is transcribed only by cells experiencing high Hh signaling levels close to the source of Hh. In wing development, rdx and the Cul3-pathway modulate the amount of Ci155 available for Cul1-dependent processing, lowering the maximum level of Ci155 activity at high Hh levels. Rdx could lower Ci155 levels in unspecified eye cells posterior to the furrow by this mechanism, in which an equilibrium between Hh-dependent induction of rdx, and rdx- and Cul3-dependent degradation of Ci155, leads to a lower level of Ci155 protein than anterior to the furrow. By contrast, in the specified, differentiating eye cells, rdx transcription becomes independent of Hh signaling, and Ci155 is degraded more completely (Baker, 2009).
If there is Hh signaling posterior to the furrow, as these studies find maintains rdx transcription in unspecified retinal cells, why are genes such as atonal that are induced by Hh signaling ahead of the furrow not also expressed posterior to the furrow? There are at least three possible explanations. First, rdx may dampen Ci155 accumulation in unspecified cells such that the threshold necessary for ato expression is not achieved posterior to the furrow. This is unlikely to be the sole explanation, since mutating rdx or cul3 permits Ci155 accumulation but does not lead to ectopic R8 specification, but it could contribute in conjunction with other mechanisms. Secondly, other genes may interfere posterior to the furrow. This could include egfr induction of Bar gene expression, since Bar genes antagonize ato expression. There seem to be multiple respects in which EGFR-dependent differentiation renders cells unable to continue anterior responses to Hh, and it is also envisaged that egfr might play a role in further mechanisms that modulate the response to Dpp signaling posterior to the furrow, should such mechanisms exist. Finally, recent evidence suggests that induction of ato by Hh is not so simple as the induction of a target gene above a threshold in a morphogen gradient, but depends indirectly on Hh repressing Eyeless and activating Sine Oculis, so that these transcription factors are coexpressed and turn on ato only in a domain ahead of the furrow. In this case, persistent Hh signaling would not be expected to activate ato expression once Ey had been repressed (Baker, 2009).
Recently, Hh has been discovered to induce compensatory proliferation in response to eye disc cell death, a further example of post-furrow Hh function. The current results now suggest the model that loss of EGFR-dependent rdx expression elevates Ci155 locally to permit Hh responses when photoreceptor cells that secrete EGFR ligands are lost. Consistent with this idea, loss of rdx or cul3 also result in proliferation of eye disc cells (Baker, 2009).
The regulation of rdx expression and thus degradation of Ci by Cullin-3 may not be sufficient to explain Ci regulation posterior to the furrow. In order for Ci155 to be stable, as observed in cul3 mutant clones and egfr mutant clones, Ci155 must escape processing to Ci75 by Cul-1. Ahead of the furrow, and in most other tissues, rdx is not expressed, Ci is not coupled to Cul3, and Ci155 is stabilized wherever Hh inhibits Smo and the Cul1 pathway. The observation that Ci155 is stable in cul3 clones, or in the genotypes where rdx is not expressed, shows that Ci155 escapes processing by the Cul1 pathway in the posterior eye as well, but this is not due to Hh. Ci155 accumulates in smo egfr mutant clones that do not express rdx and cannot respond to Hh (Baker, 2009).
One model would be that once rdx is induced, Ci155 is sequestered and not available to be processed by Cul1. This model cannot explain why Ci155 accumulates in egfr clones that lack rdx expression, where Ci155 should be available for Cul1. Therefore Ci155 must escape Cul1-mediated processing in the posterior eye by a distinct mechanism. This could be explained by the induction of a component distinct from Rdx that inhibits the processing of Ci155 by Cul1, or sequesters Ci155. It is equally possible that a component essential for processing of Ci155 by Cul1 is repressed posterior to the morphogenetic furrow (Baker, 2009).
Previous studies show that Ci155 never accumulates in smo tkv clones that are unable to respond to either Hh or Dpp signaling. Clones of cells unable to respond to Dpp, but able to respond to Hh and Ras, show only a subtle change in Ci155 labeling. These previously published observations suggest that Ci155 remains a target of Cul1 in the absence of both Dpp and Hh signaling, perhaps through failure to transcribe or repress transcription of a gene that modulates Ci155 proteolysis by Cul1 posterior to the furrow (Baker, 2009).
It is now possible to account for why smo clones affect Ci155 levels differently from cul3 clones, a previously puzzling observation. In cul3 clones, or egfr clones that do not express rdx, the Cul3 pathway cannot degrade Ci155 and the Cul1 pathway is inactivated posterior to the furrow exactly as in wild type discs, so Ci155 accumulates. In smo clones, Ci155 transiently accumulates in those cells in which processing by Cul1 has been lost but rdx not yet induced. In such cells, Ci155 is not coupled to any cullin, and is stable. Eventually, differentiation spreads into the posterior of smo clones, leading to rdx expression, and Cul3-dependent Ci degradation. If differentiation and rdx expression are prevented, as in smo egfr clones, then Ci155 remains stable. Because there is a delay in expressing rdx in smo clones compared to wildtype, Ci155 is not subject to Cul3-mediated processing as soon as in wild type, and there is a period when Ci155 has been uncoupled from Cul1-processing but not yet coupled to the Cul3 pathway. It is during this period that Ci155 accumulates in smo mutant cells (Baker, 2009).
These findings help explain how a wave of differentiation moves across the eye disc uni-directionally. Hh, secreted from differentiating photoreceptor cells, must be present at highest concentrations posterior to the furrow. Indeed, ahead of the furrow Ci155 is stabilized in a decreasing posterior-to-anterior gradient, consistent with a gradient of Hh protein coming from a source posterior to the furrow. Yet, the cell-autonomous responses to Hh signaling that are seen ahead of the furrow, such as cell cycle arrest and atonal expression, do not occur posterior to the furrow, where Ci is rendered unstable by Rdx and Cul3, induced both directly by Hh itself, and indirectly by the photoreceptor differentiation that is largely induced by EGFR posterior to the furrow (Baker, 2009).
There are other examples where Hh-secreting tissues are not the targets of Hh signaling. For, example, in Drosophila wing development, anterior compartments respond to Hh secreted by posterior compartments, but posterior compartment cells do not respond because ci transcription is repressed by the posterior-specific protein Engrailed. In vertebrate development, notochord cells express Shh but the responses seen in the nearby spinal cord are not seen in notochord. Such segregation of Hh-producing cells from fields competent to respond to Hh makes sense, if the purpose of Hh signaling in development is to pattern new body regions. Hh signaling is also deregulated in many tumors. Whether any of these tumors activate Hh signaling by affecting GLI protein stability, or other normal down-regulatory mechanisms, remains to be seen. In any case, mechanisms that render cells unresponsive to Hh by coupling Ci155 to the proteosome might prove useful in the treatment of cancers that depend on Hh signaling (Baker, 2009).
gft is identified by seven alleles, all of which are zygotic lethal and dominantly suppress the Gαs* phenotype. The ability of all gft alleles to modify the Gαs phenotype indicates that the suppression of ectopic Gαs signalling is a common property of gft mutations and likely results from a loss of gft function. All gft alleles are zygotic lethal, however, homozygous mutant embryos hatch into first instar larvae. To investigate the strength of each allele the lethal phase was determined of all alleles except gftP34, which contains a closely linked mutation in Gli (35D3) and gftGR18, which resides in a translocation background. Homozygous gftHG39, gftHG43, gftPZ06340 or gftd577 embryos or embryos transheterozygous for each allele over deficiency TE35D-GW15, hatch but die during the second larval instar. Larvae homozygous for gftB14 survive the second larval instar and die as third instar larvae prior to wandering. These data identify the earliest lethal phase of dCul-3 as the second larval instar (Mistry, 2004).
Precise excision of the P-element insertion in gftPZ06430 reverted its ability to modify the Gαs* phenotype, indicating that the P-element insertion suppresses the Gαs* phenotype and reduces gft function. The P-element insertion in gftPZ06430 was used to clone gft. Genomic DNA flanking this P-element was used to identify eight overlapping cDNAs from this region (Mistry, 2004).
To verify that gft corresponds to dCul-3, molecular lesions in all gft alleles were sought. gftB14contains a C to T transversion at nucleotide position 2129, which converts an evolutionarily conserved alanine at position 710 to a valine. l(2)gftP34 contains an in-frame deletion at nucleotide positions 615-920 that removes 101 amino acids between residues 206 and 307. l(2)gftGR18 was induced by γ-ray mutagenesis in a T(Y;2); Dp(1;2) background (Y,21-35B1; 35B2-60,Y). This lesion results in the loss of a single nucleotide at position 475, causing a frameshift at amino acid 158 and a premature stop at amino acid 167. l(2)gftPZ06430 contains a 16kb PZ element (P ry+, lacZ ) in the first intron of gft, 228 nucleotides from exon 2. gftHG39 contains a 5 nucleotide deletion at nucleotide positions 2310-2314 that results in a frameshift at amino acid 747 and a premature stop codon at amino acid 748 that removes the C-terminal 26 amino acids of the protein which comprise 50% of the Cullin motif. The lethality of a dCul-3 allele that lacks 50% of this domain provides strong evidence that the conserved C-terminus is essential for function. The identification of molecular lesions in dCul-3 in five gft alleles provides compelling evidence that gft corresponds to dCul-3. For simplicity, gft is referred to as dCul-3 (Mistry, 2004).
The large maternal contribution of dCul-3 mRNA, together with the larval lethality of dCul-3 alleles suggests that maternal dCul-3 products are sufficient for embryonic development. The requirement of dCul-3 function was examined during embryonic development by creating germ line clones that lack maternal and zygotic dCul-3 contribution using the l(2)gftPZ6340 and l(2)gftHG39 alleles. For both alleles, few eggs were recovered. These eggs were approximately half the size of wild-type and contained fused dorsal appendages, a phenotype similar to that of loss of EGF-receptor activity. No discernible embryonic structure interior to the chorion was detected. These data indicate that dCul-3 is required in the maternal germ line during oogenesis for patterning and development of the egg (Mistry, 2004).
Since mutations in dCul-3 have no obvious zygotic phenotype and embryos lacking maternal dCul-3 contribution fail to develop, mitotic clonal analysis was used to study the developmental role of dCul-3. In these studies l(2)gftPZ06430 and l(2)gftHG39 were used because these belong to the strongest class of dCul-3 alleles. Clones of these alleles exhibit identical phenotypes that can be grouped into three classes. The first phenotypic class is identified by patterning defects in the wing and notum, including alterations in their shape and size, as well as defects in the formation and position of specific cellular structures such as veins. The second class is identified by ectopic formation of sensory organs as well as defects in sensory organ cell lineage. The third class is identified by defects in cell growth and survival (Mistry, 2004).
Large dCul-3 clones exhibit a number of specific defects in adult wings, including perturbation of the overall shape of the wing and in wing vein position and formation. For example, clones that cover the L3 vein often cause a posterior shift in its position. As L3 runs immediately anterior to the AP boundary, alterations to its position likely reflect modifications to the location of the AP boundary. In addition, clones that cover L3 and other veins are associated with loss of vein tissue . Thus, dCul-3 is required to promote wing growth and patterning as well as wing vein formation, suggesting dCul-3 regulates one or more of the signalling pathways that control these events (Mistry, 2004).
dCul-3 clones in the wing and notum also contain ectopic sensory organ formation. In wild-type wings three campaniform sensillae arise evenly spaced along L3. However, dCul-3 clones that encompass a significant portion of L3 invariably contain more than three and can contain as many as eight campaniform sensillae. Ectopic campaniform sensillae also arise between L2 and the anterior wing margin and ectopic bristles arise distally between L2 and L3. dCul-3 notal clones are associated with significant tufting of both micro- and macro-chaetae. These tufts are made up of ectopic fully formed external sensory organs as well as individual sensory organs that contain multiple shafts within a single socket. The presence of ectopic fully-formed sensory organs suggests dCul-3 helps regulate the initial decision of cells to acquire the neural fate while the presence of multiple shafts within a single socket indicates dCul-3 controls cell-fate decisions in the sensory organ lineage. Reduction in Notch pathway function also promotes ectopic sensory organ formation and shaft duplications, hinting at a possible link between dCul-3 and Notch signalling activity (Mistry, 2004).
This study investigated whether dCul-3 overexpression produces reciprocal wing and notal phenotypes relative to loss of dCul-3 function. Overexpression of full-length dCul-3 (dCul-3FL) along the AP boundary results in decreased intervein territory between L3 and L4, partial or complete loss of the L3 campaniform sensillae and ectopic vein formation. Similarly, overexpression of dCul-3FL in all proneural clusters using scabrous-GAL4 (sca-GAL4) causes a severe loss of macrochaetae throughout the notum as well as campaniform sensillae and anterior margin bristles in the wing. Thus, with respect to vein formation and sensory organ development, overexpression of dCul-3FL results in reciprocal phenotypes to those observed in dCul-3 mutant clones (Mistry, 2004).
Studies in other systems have provided contradictory results on the importance of the Cullin C-terminal domain. Identification of a mutation in the dCul-3 C-terminal domain that severely disrupts dCul-3 function supports the importance of this highly conserved domain. To address this issue further, a truncated form of dCul-3 that specifically lacks the C-terminal domain was driven under the control of sca-GAL4 or dpp-GAL4. Overexpression of this protein has no effect on wing development. These data underline the importance of the C-terminal domain for dCul-3 function (Mistry, 2004).
Perturbation of dCul-3 in proneural clusters affects sensory organ formation. Genetic studies indicate that dCul-3 function opposes sensory organ development. To investigate when dCul-3 exerts its effect on sensory organ development, the expression of neuralized-LacZ (neurLacZ) was followed in mitotic clones of dCul-3 in wing imaginal discs. neurLacZ is expressed in all sensory organ precursors (SOPs) of developing third instar imaginal discs. dCul-3 mitotic clones that overlap areas of SOP formation show a dramatic increase in SOP numbers, while clones that do not overlap areas of SOP formation appear wild-type. Therefore, dCul-3 inhibits SOP development and dCul-3 might normally act either to stabilise proteins that promote sensory organ development, such as Achaete or Scute, or to inhibit proteins that oppose sensory organ development such as members of the Notch pathway (Mistry, 2004).
To investigate if dCul-3 overexpression inhibits SOP formation, Achaete (Ac) and Cut expression was followed in wing imaginal discs in which dCul-3FL was over-expressed using dpp-GAL4 and sca-GAL4. Ac is expressed in proneural clusters and promotes SOP formation while SOPs activate Cut shortly after they form. Overexpression of dCul-3FL leads to the absence or severe reduction of Ac expression in proneural clusters. For example, dCul-3FL overexpression along the AP compartment boundary causes a severe reduction in both Ac and Cut expression in the L3 cluster and a reduction of Ac expression in part of the dorsal component of the anterior wing margin. Overexpression of dCul-3FL in all proneural clusters using sca-Gal4 reduces Ac expression in the anterior wing margin and L3 cluster and also reduces or eliminates Ac expression in most proneural clusters in the notum which in turn results in the loss of SOPs and macrochaetae. These data indicate that dCul-3 affects sensory organ formation by inhibiting Ac expression and thus limiting the ability of cells to acquire the sensory organ precursor fate (Mistry, 2004).
Many of the dCul-3 wing and notal phenotypes are similar to those that arise due to defects in different developmental signalling pathways. Consistent with dCul-3 regulating Hh-pathway activity, dCul-3 clones in third instar eye imaginal discs exhibit a cell-autonomous accumulation of Ci155 posterior to the morphogenetic furrow, but have no effect on Ci155 accumulation anterior to the morphogenetic furrow. Ou (2002) has reported similar observations in dCul-3 eye clones. Because of the importance of targeted proteolysis in the Hh pathway and the roles the Hh pathway plays in setting up the AP boundary of the wing as well as the location of the L3 and L4 veins, whether dCul-3 modulates Hh activity in wing imaginal discs was investigated. Active Hh signalling prevents proteolysis of the full-length form of Ci (Ci155), the transcriptional effector of the Hh pathway. Therefore, an antibody specific to Ci155 was used to assay whether dCul-3 function affects Hh signalling by regulating the stability of Ci155 in larval third instar wing imaginal discs (Mistry, 2004).
Normally, Ci155 is expressed throughout the anterior compartment of the wing disc but accumulates to higher levels in a stripe at the AP boundary in response to high levels of Hh. dCul-3 clones that arise anywhere in the anterior compartment exhibit no clear effect on Ci155 accumulation. Similarly, clones that arise in the posterior compartment and at a distance from the AP boundary have no effect on Ci155. However, dCul-3 clones that reside in the posterior compartment and abut the AP boundary appear to cause a moderate non-autonomous reduction of Ci155 accumulation in cells of the anterior compartment immediately adjacent to the clone. Since high level Ci155 accumulation in these cells normally requires transmission of the Hh signal from posterior compartment cells, these data suggest that dCul-3 activity might modulate transmission of the Hh signal. Thus, in both wing and eye imaginal discs dCul-3 function regulates Ci155 accumulation: in the wing, loss of dCul-3 results in a non-autonomous decrease of Ci155 accumulation, while in the eye, loss of dCul-3 results in a cell autonomous accumulation of Ci155 (Mistry, 2004).
Given the effect loss of dCul-3 function has on Ci155 accumulation, it was asked if dCul-3 overexpression modulates Ci155 accumulation. sca-GAL4 was used to overexpress dCul-3FL in all proneural clusters of the wing imaginal disc and along the entire dorsal-ventral (DV) boundary of the wing margin and a cell-autonomous accumulation of Ci155 was observed in all Sca-expressing cells in the anterior compartment. No effect on Ci155 accumulation in the posterior compartment was observed even though dCul-3FL is overexpressed in the posterior domain of the wing blad. Similarly, dCul-3 overexpression in the leg and haltere, but not eye, results in a cell-autonomous accumulation of Ci155 in the anterior but not posterior compartment. Overexpression of dCul-3 in the wing, leg and haltere imaginal discs using other GAL4 driver lines, results in an identical cell-autonomous accumulation of Ci155 in the anterior compartment. Thus, overexpression of dCul-3FL acts cell-autonomously and specifically in the anterior compartment to promote high-level accumulation of Ci155. These results suggest that dCul-3 directly opposes the action of a protein required to promote Ci cleavage, or that it exhibits dominant negative activity by titrating other SCF components required for Ci proteolysis, such as dCul- (Mistry, 2004).
Costal-2 (Cos-2), Fused (Fu) and Suppressor of Fused (Su(Fu)) form a complex with Ci155 and regulate its cleavage. Antibodies against Cos-2, Fu and Su(Fu) were used to investigate if these proteins are also overexpressed in scaGal4::UASdCul-3FL wing imaginal discs. In contrast to Ci155, expression of all three proteins appears normal in when scaGal4 was used to drive UASdCul-3FL in wing imaginal discs suggesting that overexpression of dCul-3FL specifically targets Ci (Mistry, 2004).
To examine whether persistent dCul-3 overexpression in the posterior compartment might lead to heightened Ci155 accumulation, en-Gal4 was used to overexpress dCul-3FL in this domain. Overexpression of dCul-3FL in the posterior compartment causes a drastic reduction in the size of this compartment and a concomitant increase in the size of the anterior compartment. The drastic size reduction of the posterior compartment along with defects in DV boundary formation in the posterior compartment indicate that overexpression of dCul-3FL leads to defects in cell-survival and cell-fate specification in this domain (Mistry, 2004).
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date revised: 10 October 2011
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