supernumerary limbs


Effects of Mutation or Deletion

In the Drosophila leg disc, wingless and decapentaplegic are expressed in a ventral-anterior and a dorsal-anterior stripe of cells, respectively. This pattern of expression is essential for proper limb development. While the hedgehog (Hh) pathway regulates dpp and wg expression in the anterior-posterior (A/P) axis, mechanisms specifying their expression in the dorsal-ventral (D/V) axis are not well understood. Evidence is presented that slimb mutant clones in the disc deregulate wg and dpp expression in the D/V axis. This suggests for the first time that their expression in the D/V axis is actively regulated during imaginal disc development. Furthermore, slimb is unique in that it also deregulates wg and dpp in the A/P axis. The misexpression phenotypes of slimb- clones indicate that the regulation of wg and dpp expression is coordinated in both axes, and that slimb plays an essential role in integrating A/P and D/V signals for proper patterning during development. Genetic analysis further reveals that slimb intersects the A/P pathway upstream of smoothened (smo) (Theodosiou, 1998).

slimb was identified in a mutant screen. To identify recessive overproliferation mutations in genes that are lethal in homozygous mutant animals, genetic screens were performed in mosaic flies containing homozygous mutant patches in otherwise wild-type backgrounds. Two classes of recessive overproliferation mutations have been identified. Mutations of the first group cause mutant cells to undergo extensive proliferation and form unpatterned, tumorous outgrowths in mosaic adults. Mutations of the second group induce both patterned and irregular outgrowths. slimb affects developmental signals that regulate cell proliferation and pattern organization. The slimb transcript encodes a Cdc4-related protein containing F-box and WD-40 motifs. Jiang (1998) has independently reported the identification of this gene. Using a Drosophila slimb cDNA, a human homolog (H-slimb) has been isolated. The fly and human proteins share 78% amino acid identity throughout, suggesting that slimb is functionally conserved (Theodosiou, 1998).

slimb-induced outgrowths are reminiscent of the phenotypes caused by misexpression of dpp and wg. dpp and wg expression were examined in slimb mosaic leg discs using wg-lacZ and dpp-lacZ reporter genes. slimb clones ectopically express both wg and dpp in a cell-autonomous fashion. slimb mutant clones deregulate wg and dpp in both D/V and A/P axes. Ectopic wg expression is observed in both ventral and dorsal regions. Similar results are also observed for dpp. In slimb mutant clones situated within or near the endogenous dpp expression zone, dpp is expressed in the mutant cells but down-regulated in adjacent wild-type cells. Previously it had been shown that Wg and Dpp signaling mutually antagonize each other's expression, which prevents expression of the two molecules in the same cells. Ectopic expression of both wg and dpp in slimb- clones in the dorsal-anterior of the leg disc indicates a disruption of this mutual antagonism. To test whether ectopic wg and dpp expression are responsible for the outgrowth phenotype in slimb mosaic animals, flies were generated carrying clones of cells mutant for both slimb and wg, or slimb and dpp. In comparison to slimb mutant clones, double mutant clones do not cause any significant outgrowths. Therefore, Wg and Dpp are two primary effector molecules responsible for the induction of outgrowths in slimb mosaic animals. These results are consistent with previous observations that wg and dpp are both required for defining the proximodistal outgrowth center (Theodosiou, 1998).

The slimb phenotype differs from those of all previously known genes, in that it is the first gene found to deregulate both wg and dpp expression in the D/V axis. Disrupting components of the Hh signaling pathway deregulate wg and dpp only along the A/P axis. Thus, the control of wg and dpp expression in the D/V axis is not disrupted by disruption of the Hh pathway. The mechanism restricting wg and dpp in the D/V axis is not known. The mutant phenotype of slimb- clones in discs provides the first evidence that wg and dpp expression in the D/V axis is actively regulated during imaginal disc development, and is not solely defined during embryonic development. Since the Hh pathway regulates wg and dpp expression in the A/P axis, these results suggest that a pathway different from Hh may operate in imaginal discs to restrict their expression in the D/V axis. This pathway cannot be either the Wg or Dpp signaling pathway since inactivation of Wg or Dpp signaling is known to affect either dpp or wg expression, but not both. The slimb phenotypes described here were not observed in the previous study which used weak slimb alleles and revealed only A/P defects (Jiang, 1998). Jiang proposed that Slimb protein normally targets Ci and Arm for processing or degradation by the ubiquitin/proteasome pathway, and that Hh and Wg regulate gene expression, at least in part, by inducing changes in Ci and Arm, which protect both Ci and Arm from Slimb-mediated proteolysis. The phenotypic differences probably reflect the fact that a null allele was used in the current study instead of hypermorphic alleles. In addition to D/V defects, slimb mutant clones also deregulate wg and dpp expression in the A/P axis. slimb is the first identified gene that regulates both wg and dpp expression in the A/P as well as D/V axes (Theodosiou, 1998).

To further explore how slimb regulation and function correlates with A/P signaling, double mutant analysis was carried out with slimb mutants and with mutants of hh and smo. No reduction of outgrowths was observed in slimb-, hh- double mutant clones. Furthermore, slimb mutant clones have no effect on hh expression. This indicates that slimb acts downstream or independent of Hh signaling. In contrast, slimb-, smo- double mutant clones almost completely suppress slimb induced outgrowths. Consistent with the adult phenotype, discs carrying slimb- , smo- clones fail to ectopically express either dpp or wg. These data suggest that slimb intersects the A/P signal upstream of smo. Jiang (1998) suggested that slimb acts downstream of smo. This difference may be explained by the use of different alleles for smo and slimb. The Slimb product contains WD-40 repeats believed to act as a scaffold for the binding of multiple proteins. It is possible that this structure may allow for proteins such as Smo and components of a D/V pathway to converge. The Slimb-related protein Cdc4 from Saccharomyces cerevisiae along with Cdc53, and Cdc34 are part of the ubiquitin proteolysis machinery. The current data that Slimb acts upstream of Smo, together with its sequence homology with Cdc4, suggests that Slimb could be involved in the regulation of Smo protein degradation (Theodosiou, 1998).

Slimb (Slmb) is an F-box/WD40 protein that potentially participates in the ubiquitin proteolysis machinery. During development, Slmb is required in limb discs to repress Hedgehog (Hh) target genes, i.e. wingless and decapentaplegic, as well as the Wg signal transduction pathway. These repression functions have been proposed from studies using weak slmb alleles. Interestingly, experiments with strong slmb alleles have revealed additional functions in which slmb is required, such as leg dorsal-ventral restriction. New alleles of the slmb gene have been isolated in a screen for new negative regulators of dpp: several amorphs (characterized by genetic and molecular criteria) and a cold-sensitive hypomorph. By performing somatic clone experiments with these new amorphic slmb alleles, it has been determined that regulation of Dpp and Wg by Slmb could be different from what has already been published. In leg discs, lack of slmb function derepresses the transcription of wg, independent of Hh signaling. Ectopic legs resulting from slmb- clone induction come only from wg misexpression in the normal dpp domain, since ectopic proximo-distal axes are induced dorsally, and adult ectopic legs are often perfect with respect to antero-posterior polarity. In wing discs, transcription of wg, which is normally independent of Hh signaling, is also derepressed in the absence of slmb function. In discs bearing amorphic slmb clones and in discs of two other slmb- contexts, a novel pattern of dpp expression is described consisting of an enlargement of the normal dpp domain. Strong evidence indicates that this dpp modification can be linked to imaginal disc regeneration following slmb- cell elimination. The fate of slmb- clones, which disappear before adulthood, has been investigated. It was found that two mechanisms of cell elimination can account for imaginal cell regeneration: an early apoptosis and a mechanism of sorting-out that excludes all slmb- clones from all imaginal discs. This result suggests that Slmb is likely to be involved, in addition to its repression role on Dpp and Wg, in some other essential cellular mechanism, since, in the absence of Slmb, cell affinities are dramatically modified regardless of the deregulated morphogen and of the type of imaginal disc (Miletich, 2000).

These results indicate that slmb is involved in the repression of wg transcription independent of Hh signaling. In the wing pouch, wg transcription depends on communications between dorsal and ventral cells involving the Notch receptor. This finding suggests that slmb is involved in the proteasome-dependent degradation or proteolytic cleavage of a putative regulatory protein of the Notch signaling pathway. This is in good agreement with a proposal that the proteasome is involved in the degradation of an active form of Notch, thus limiting the activation of the Notch targets. In the leg disc, the mechanism responsible for wg transcription in the posterior cells is unknown. In the anterior compartment of imaginal discs, the transcription factor Ci is necessary to activate (or repress) Hh target genes. In the posterior compartment cells, engrailed represses transcription of ci. Thus, a deregulation of wg transcription depending on Hh signaling must be linked to ectopic ci transcription. Ectopic transcription of ci is not observed in the posterior compartment of leg discs bearing slmb- clones; neither is a switching-off of en transcription observed. It is concluded that a repression of wg transcription occurs in leg discs irrespective of the Hh/Ci signaling, and that slmb is involved in this process (Miletich, 2000).

In conclusion, two mechanisms appear to eliminate slmb- cells in imaginal discs: an early apoptosis that only concerns some cells, and a mechanism for sorting-out that excludes all slmb- clones from all imaginal tissues. The early apoptosis is possibly induced by differential cell adhesion: in this case, it would be classified as an early sorting-out. Another possibility is that apoptosis is an alternative path to sorting-out when mutant cells form a 2-4 cell group (the theoretical size of a clone aged 24 h) rather than an organized population of cells (as found 48 h after clone induction). This could be answered by investigating whether early apoptosis is causally linked to a modification of cell affinity. It is also important to investigate whether slmb plays a direct role in the appearance of apoptotic cell death. If so, slmb would act as an anti-apoptotic gene; preliminary results favoring this result have been obtained by generating slmb- clones in a context of apoptosis inhibition (Miletich, 2000).

An important feature of the disappearance of null slmb- clones is that sorting-out occurs regardless of the deregulated signaling pathway and of the type of imaginal disc. Therefore, it seems that this exclusion is not a result of the deregulation of these pathways but rather is the result of the deregulation of some other essential cellular mechanism shared by all imaginal cells. Since slmb encodes an F-box protein that would be involved in targeting degradation of proteins by the proteasome, it is proposed that slmb is necessary in a general process required for the proper functioning of many cellular mechanisms. Alteration of this process would then lead to such dramatic changes in cell affinities that all slmb- cells would be excluded from all types of imaginal discs (Miletich, 2000).

The duplication of the centrosome is a key event in the cell-division cycle. Although defects in centrosome duplication are thought to contribute to genomic instability and are a hallmark of certain transformed cells and human cancer, the mechanism responsible for centrosome duplication is not understood. Recent experiments have established that centrosome duplication requires the activity of cyclin-dependent kinase 2 (Cdk2) and cyclins E and A. The stability of cyclin E is regulated by the ubiquitin ligase SCF, which is a protein complex composed of Skp1, Cdc53 (Cullin) and F-box proteins. The Skp1 and Cullin components have been detected on mammalian centrosomes, and shown to be essential for centrosome duplication and separation in Xenopus. Slimb, an F-box protein that targets proteins to the SCF complex, plays a role in limiting centrosome replication. In Drosophila the hypomorphic mutation slimbcrd causes the appearance of additional centrosomes and mitotic defects in mutant larval neuroblasts (Wojcik, 2000).

A mutant Drosophila line, initially named centrosome replication defective (crd), was detected in a screen of late larval and pupal lethal mutants from a collection of third chromosome P-element insertion mutants. Examination of larval neuroblast chromosome spreads revealed that homozygous crd mutants display two types of abnormal mitotic figures: metaphase figures comprising overcondensed chromosomes, and polyploid figures suggestive of defects in progressing through the mitotic cycle. The single P element in this line maps to position 93B10-13 by in situ hybridization to polytene chromosomes. The chromosomal deficiency Df(3R)eR1 uncovered the crd mutation and the resulting hemizygous animals also have a mitotic phenotype similar to the homozygous crd mutants. Whereas crd homozygotes die at the larval-pupal boundary, the mitotic defects and lethality of crd were reverted by excision of the P element (Wojcik, 2000).

Isolation of genomic DNA flanking the crd P-element insertion revealed that it is inserted 296 bp into the 5' untranslated region (UTR) of a previously identified locus, supernumerary limbs (slimb). Genetic analysis confirmed that crd and the slimb mutations are allelic and responsible for the observed centrosome replication defect. Accordingly, the allele was renamed slimbcrd (Wojcik, 2000).

To characterize further the slimbcrd mitotic phenotype, the morphology of hemizygous slimbcrd neuroblasts was examined by confocal microscopy. Most striking of the abnormalities in mutant mitotic cells was the excessive number of centrosomes revealed by the distribution of the centrosomal antigen CP190. Diploid cells were observed that contained more than two and as many as seventeen centrosomes. Polyploid giant neuroblasts were also observed in mutant brain discs, containing far greater numbers of centrosomes than predicted by a failure in cleavage alone (Wojcik, 2000).

To confirm that the CP190-positive structures identify centrosomes, tests were performed for the presence of the majority of the known intrinsic components of Drosophila microtubule-organizing centers, including gamma-tubulin, Centrosomin (CNN) and Abnormal spindle (ASP). Each component was present at the putative centrosomes. Double-label experiments using antibodies against gamma-tubulin, CP190, CNN or ASP showed that signals from all these antigens always coincide with the putative centrosome foci in both wild-type and slimbcrd mutant mitotic neuroblasts. In addition, the size and shape of the multiple foci in slimbcrd neuroblasts are uniform and comparable to centrosomes in wild-type cells. Taken together, these observations are most consistent with the presence of excess numbers of centrosomes in diploid slimbcrd mutant cells, and are not readily explained by the aberrant aggregation of centrosomal antigens or the fragmentation of a single pair of centrosomes (Wojcik, 2000).

Analysis of centrosome number in mutant diploid neuroblasts revealed that a majority of cells (66%) contained excess centrosomes. The aberrant number of centrosomes often exceeds the four foci that would be expected if the defect arose from the precocious separation of the centriole pair associated with each centrosome. Instead, the aberrant centrosome numbers suggest that repeated rounds of centrosome duplication occur during individual cell cycles in slimbcrd cells. Significantly, the increase in the number of centrosomes is not random, with 74% of cells, excluding normal cells with two centrosomes, containing even numbers of centrosomes. This result suggests that not all of the extant centrosomes are licensed to replicate, but is more consistent with the continuous replication of the starting pair of centrosomes. This phenotype is distinct from an assembly/fragmentation defect in centrosome morphogenesis recently reported for a Drosophila Hsp90 mutation (Wojcik, 2000).

It is striking that, despite the presence of excess centrosomes in slimbcrd cells, the spindles are bipolar and show no indication of branching or multipolarity as seen in other mitotic mutants. For centrosomes considerably displaced from the poles, no substantial or stable microtubule-organizing activity was apparent, further suggesting that the additional centrosomes are 'immature' or functionally distinct. Still, the low incidence of hyperploid cells associated with the slimb mutations suggests that, despite the lack of severe spindle defects, excess centrosomes may disrupt the downstream events associated with cytokinesis or cleavage (Wojcik, 2000).

The failure of homozygous slimb- clones to proliferate in mosaic animals is consistent with the cell-cycle defects observed. In Drosophila embryos, Xenopus and sea-urchin extracts, as well as mammalian cells, centrosome duplication is closely tied to known mitotic regulators. Therefore, it is possible that slimb mutations affect centrosome replication indirectly by generally modulating progression through mitosis. Because it has previously been shown that centrosome replication is coupled to S phase in vitro, and that an abnormally prolonged S phase can result in centrosome over-replication, an examination was made to determine whether the duration of S phase was increased in slimbcrd cells. If S phase is lengthened in the slimb mutant cells, then an increase in the total number of S-phase cells seen at any given moment in mutant, compared with wild-type, tissues, would be expected. Instead, a decrease was found in the number of S-phase cells in slimb mutant brains. Therefore, it is proposed that Slimb is not likely to lengthen the progression of mitosis and, instead, acts more directly to stop centrosome replication (Wojcik, 2000).

The slimb gene was first identified as a negative regulator of the Hedgehog (Hh) and Wingless (Wnt/Wg) signaling pathways in Drosophila. It was recognized as a member of the F box/WD40 class of proteins that can act as targeting factors for the SCF complex, an E3 ubiquitin ligase first identified from cell-cycle studies in yeast. SCF activity is also required to degrade cell-cycle regulatory proteins in metazoans, as is evident from the accumulation of cyclin E in mouse knockouts deficient for its Cullin 1 component. In Drosophila neuroblasts, Slimb is required to restrict centrosome duplication during the cell cycle. The target whose presumed degradation is regulated by Slimb is not known. One possibility is that the target is cyclin E, since this Cdk2 subunit is known to be degraded by the SCF complex and is also required for centrosome duplication. This may in part explain why antibodies to either the Skp1 or Cullin1 components of SCF can block the initial separation of replicating centrioles in vitro. Nevertheless, because individual F-box proteins, such as Slimb, can interact with more than one target protein, and multiple F-box proteins localize at centrosomes during mitosis, other centrosomal targets and distinct steps in centrosome replication are likely to be involved (Wojcik, 2000).

The requirement for an E3 ubiquitin ligase targeting component to regulate both signaling pathways and centrosome duplication offers one means of coordinating the regulation of developmental processes, and signals for cell proliferation, with the mechanics of cell-cycle progression. It is not difficult to imagine how competition for rate-limiting levels of Slimb protein might regulate the division of cells within a particular developmental program. Similar regulatory networks may also be relevant in the significant number of human cancers in which the degradation of ß-catenin is dysregulated and in human tumor cells with the known occurrence of excess centrosomes (Wojcik, 2000).

Photoreceptor differentiation in the Drosophila eye disc progresses from posterior to anterior in a wave driven by the Hedgehog and Decapentaplegic signals. Cells mutant for the hyperplastic discs gene misexpress both of these signaling molecules in anterior regions of the disc, leading to premature photoreceptor differentiation and overgrowth of surrounding tissue. hyperplastic discs encodes a HECT domain E3 ubiquitin ligase that is likely to act by targeting Cubitus interruptus and an unknown activator of hedgehog expression for proteolysis (Lee, 2002).

If hyd regulates dpp expression by altering Ci activity, loss of hyd should lead to upregulation of full-length, active Ci. Increased levels of full-length Ci are indeed observed in hyd mutant clones in the anterior of the eye disc. However, this could be due to misexpression of hh in the same clones. To determine whether hyd has a direct effect on Ci, hyd;hh double mutant clones anterior to the morphogenetic furrow were examined. High levels of full-length Ci accumulated in these clones, confirming that Hyd normally reduces Ci levels independently of Hh activity (Lee, 2002).

The F-box protein Slmb has been shown to promote processing of Ci to Ci75 as a component of an SCF ubiquitin ligase complex. Therefore the effects were compared of slmb and hyd mutations on Ci levels in the wing disc. Ci155 is much more dramatically increased in slmb clones than in hyd clones. An interesting difference was also observed between hyd and slmb in the regulation of dpp. dpp expression is increased in hyd mutant clones close to the AP border, but is very rarely activated outside this domain. In contrast, slmb mutant clones activated dpp expression only when they lay outside the wing pouch, perhaps because of activation of Wg signaling, which represses dpp expression, within the wing pouch. Consistent with these third instar phenotypes, anterior duplications like those resulting from loss of slmb are not observed in adult wings containing hyd mutant clones, although outgrowths did arise from internal regions of the wing. Such duplications would require dpp to be misexpressed at a distance from its normal domain of expression. Ptc expression, which requires activation of the full-length form of Ci, was not altered in either hyd or slmb mutant clones. Slmb and Hyd thus appear to have distinct effects on Ci protein accumulation and activity, suggesting that they have either different substrates or different effects on the same substrate (Lee, 2002).

Cullins are the major components of a series of multimeric ubiquitin ligases that control the degradation of a broad range of proteins. The ubiquitin-like protein, Nedd8, covalently modifies members of the Cullin family. Nedd8 modifies Cullin 1 (Cul1, also known as Lin-19-like or simply Lin-19) in Drosophila. In mutants of Drosophila Nedd8 and Cul1, levels of the signal transduction effectors, Cubitus interruptus (Ci) and Armadillo, and the cell cycle regulator, Cyclin E (CycE), are unusually high, suggesting that the Cul1-based multimeric SCF ubiquitin ligase complex requires Nedd8 modification for the degradation processes of Ci, Arm, and CycE in vivo. Two distinct degradation mechanisms modulating Ci stability in the developing eye disc are separated by the morphogenetic furrow (MF) in which retinal differentiation is initiated. In cells anterior to the MF, Ci proteolytic processing promoted by PKA requires the activity of the Nedd8-modified Cul1-based SCFSlimb complex. In posterior cells, Ci degradation is controlled by a mechanism that requires the activity of Cul3, another member of the Cullin family. This posterior Ci degradation mechanism, which partially requires Nedd8 modification, is activated by Hedgehog (Hh) signaling and is PKA-independent (Ou, 2002).

The Cul1-based SCFSlimb complex controls CiFL processing in the anterior cells of the eye disc. In contrast, Cul3-mediated Ci degradation mechanism is specific to the posterior cells. These specific activities in controlling Ci protein stability are not caused by differential gene expression of Cul1 or Cul3 in the eye disc. Ubiquitous mRNA expression patterns of both Cul1 and Cul3, and ubiquitous Cul1 protein expression are found all along the eye disc, suggesting that control of specificity is mediated by mechanisms other than regulation of Cul1 and Cul3 expression (Ou, 2002).

The genetic evidence suggests that Nedd8 is directly required for CiFL proteolytic processing, consistent with the hypothesis that neddylation affects CiFL proteolysis through regulating SCFSlimb activity. Cullin proteins are the identified targets for Nedd8 modification. In the Drosophila genome, six Cullin proteins are identified, each corresponding to a mammalian homolog. Among them, Cul1 is involved in the formation of SCF complexes that function as E3 ligase. The null Cul1 allele, Cul1EX was generated. In Cul1EX homozygous larvae in the first instar stage, the Cul1 signal detected by anti-Cul1 antibodies is almost completely absent. The residual Cul1 protein in Cul1EX larvae is probably maternally contributed (Ou, 2002).

In addition to the Nedd8-Cul1 core component, the SCF complex also includes a substrate-specific F-box protein. To investigate whether SCF activity in CiFL processing is limited to the anterior cells of the eye disc, the mutant phenotype of slimb was examined. slimb is required for CiFL proteolytic processing in tissues such as wing and leg discs. When slimb1 mutant clones were generated in eye discs, high levels of CiFL accumulation were detected exclusively in clones located anterior to the MF. No accumulation of CiFL could be detected in posterior slimb1 clones. Suppression of CiFL accumulation in the posterior cells was not due to possible residual activity present in hypomorphic slimb1, because identical results of CiFL accumulation were observed in the strong hypomorphic allele slimb2 and the null allele slimbP (Ou, 2002).

In summary, the results strongly suggested that in vivo, the Nedd8-modified, Cul1-based SCFSlimb complex controls CiFL proteolysis in anterior cells. Following the sweep of the MF, CiFL stability in the posterior cells is controlled by an SCFSlimb-independent mechanism (Ou, 2002).

A ubiquitin-proteasome pathway represses the Drosophila immune deficiency signaling cascade

The inducible production of antimicrobial peptides is a major immune response in Drosophila. The genes encoding these peptides are activated by NF-κB transcription factors that are controlled by two independent signaling cascades: the Toll pathway that regulates the NF-κB homologs, Dorsal and DIF; and the IMD pathway that regulates the compound NF-κB-like protein, Relish. Although numerous components of each pathway that are required to induce antimicrobial gene expression have been identified, less is known about the mechanisms that either repress antimicrobial genes in the absence of infection or that downregulate these genes after infection. In a screen for factors that negatively regulate the IMD pathway, two partial loss-of-function mutations were isolated in the SkpA gene that constitutively induce the antibacterial peptide gene, Diptericin, a target of the IMD pathway. These mutations do not affect the systemic expression of the antifungal peptide gene, Drosomycin, a target of the Toll pathway. SkpA encodes a homolog of the yeast and human Skp1 proteins. Skp1 proteins function as subunits of SCF-E3 ubiquitin ligases that target substrates to the 26S proteasome, and mutations affecting either the Drosophila SCF components, Slimb and Cullin1, or the proteasome also induce Diptericin expression. In cultured cells, inhibition of SkpA and Slimb via RNAi increases levels of both the full-length Relish protein and the processed Rel-homology domain. It is concluded that in contrast to other NF-κB activation pathways, the Drosophila IMD pathway is repressed by the ubiquitin-proteasome system. A possible target of this proteolytic activity is the Relish transcription factor, suggesting a mechanism for NF-κB downregulation in Drosophila (Khush, 2002).

In wild-type flies Diptericin is tightly controlled by the IMD pathway. Therefore, to identify genes that normally function to repress the IMD pathway, 2,000 yellow, white (y,w) F1 male progeny from male flies mutagenized with ethyl methanesulfonate were screened for constitutive expression of a Green Fluorescent Protein (GFP) reporter gene under the control of the Diptericin promoter. Two males, J6 and G49, expressed Diptericin-GFP, and this gene was constitutively expressed in larvae and adults in homozygous lines derived from these males. Although flies carrying the J6 and G49 mutations are viable and fertile at 25°C, G49 is pupal lethal at 29°C, indicating temperature-sensitive phenotypes associated with this mutation (Khush, 2002).

Using recombination mapping, the J6 and G49 mutations were shown to be tightly linked to the y locus on the proximal tip of the X chromosome. To further localize the two mutations, deletions were used to determine that J6 falls in the area defined by the overlap of Df(1)74k24.1, Df(1)svr, and Df(1)su(s)83, placing it in cytological region 1B10 near the Dredd gene. Two lethal P-element insertions in the Bloomington stock center collection, l(1)G0389 and l(1)G0109, which map near this region, were shown to not complement the constitutive Diptericin expression in the J6 and G49 lines. By sequencing DNA flanking the P elements in the two insertion lines, both elements were ascertained to lie within 200 bp of each other in the 5' untranslated region of the SkpA gene. To confirm that J6 and G49 are mutations in SkpA, a wild-type SkpA transgene on the second chromosome was shown to suppress the constitutive Diptericin expression phenotype in G49 flies. The J6 and G49 lines were shown to each contain a point mutation in the SkpA gene that generates a single amino acid change in the SkpA protein: J6, renamed SkpAJ6, converts threonine 98 to an isoleucine, and G49, renamed SkpAG49, replaces glutamic acid 101 with a lysine. These alleles are hypomorphic mutations of SkpA since the P-element insertions are pupal lethal at 25°C. SkpAG49 is pupal lethal at 29°C, and homozygous SkpAG49 adults transferred to 29°C express Diptericin at similar levels as flies heterozygous for SkpAG49 and either the P-element insertions or deletions that remove SkpA. At 29°C, therefore, SkpAG49 behaves like a null mutation, which probably reflects the significant change from the negatively charged glutamic acid to the positively charged lysine in this allele (Khush, 2002).

The SkpA gene encodes a protein that is highly similar to Skp1 proteins in humans and yeast. Skp1 proteins are components of SCF ubiquitin ligases that target substrates to the proteasome, and crystal structures of human Skp1 complexed with the F-box protein Skp2 and the cullin protin Cul1 have been solved. SkpAJ6 and SkpAG49 both affect a conserved region of SkpA that corresponds to helix 5 of Skp1; helix 5 forms part of the core interface between Skp1, the F-box region of Skp2, and the amino-terminal domain of Cul1, with some amino acids in this helix making direct contact with residues in Skp2 and Cul1. This suggests that the SkpAJ6 and SkpAG49 mutations disrupt interactions between SkpA and the F-box protein and cullin components of an SCF complex. Protein interaction studies indicate that SkpA functions with the F-box protein Slimb and the Cullin-like protein Cullin1 (Cul1) in a Drosophila SCF complex. In support of this model, slimb1 and dcul1l(2)02074 mutant larvae, as well as larvae carrying the DTS5 mutation, a dominant-negative mutation that affects the β6 subunit of the 26S proteasome, were shown to express Diptericin at levels comparable to those in the SkpA mutants. To further test the DTS5 phenotype, the UAS-Gal4 system was used to overexpress a UAS-DTS5 transgene in larval fat bodies: DTS5 overexpression induces Diptericin to levels that are comparable to those generated by bacterial infection with Erwinia carotovora carotovora 15 (Ecc15). Flies heterozygous for mutations at both the SkpA and slimb loci were generated: these flies constitutively express Diptericin, indicating a synergistic interaction between SkpA and slimb. The constitutive Diptericin expression in the slimb1, dcul1l(2)02074, and DTS5 mutants and the interaction between SkpA and slimb together suggest that an SCFSkpA/Cul1/Slimb ubiquitin ligase represses Diptericin expression by targeting a regulatory factor for degradation by the 26S proteasome (Khush, 2002).

To determine if the constitutive Diptericin expression in the SCF complex mutants is mediated through the IMD pathway, Diptericin levels were examined in larvae homozygous for mutations in either SkpA, or slimb and various genes of the IMD pathway: SkpAJ6;imd1 and SkpAG49;dtak11 double mutants display constitutive Diptericin expression, although Diptericin levels are slightly reduced in the SkpAG49;dtak11 larvae. Mutations in DmIkkγ, DmIkkβ, and Relish, however, completely block Diptericin expression in the SkpAJ6 background, and a Dredd mutation completely blocks Diptericin expression in the slimb1 background. The constitutive Diptericin expression observed in SkpA and slimb mutants, therefore, does not require IMD and dTak1, but it is dependent on the DmIKK complex, Dredd, and Relish. These results imply that, in wild-type flies, the SCFSkpA/Cul1/Slimb negatively regulates the IMD pathway by targeting one of these factors, or an additional unidentified component of the IMD pathway, for degradation by the proteasome. In contrast to fat body cells, the IMD pathway is the primary regulator of all antimicrobial genes, including Drosomycin, in surface epithelial tissues. A Drosomycin-GFP transgene is constitutively expressed in tracheal cells but not in fat body cells of slimb1 mutant larvae; this expression pattern further demonstrates that the IMD pathway, but not the Toll pathway, is constitutively activated when the SCFSkpA/Cul1/Slimb complex is compromised (Khush, 2002).

Although the genetic results do not allow differentiating between the DmIKK complex, Dredd, Relish, or other unidentified downstream components of the IMD pathway as targets of the ubiquitin-proteasome pathway, the mammalian Relish homolog, P105, is regulated by an SCF complex that contains the Slimb homolog β-TrCP/E3RSIκB. Consequently, RNA-mediated interference (RNAi), an effective technique for specifically inhibiting targeted proteins, was used in cultured Drosophila S2 cells to test for interactions between the SCFSkpA/Cul1/Slimb complex and Relish. Initially, SkpA and Slimb activity were blocked in S2 cells via RNAi; then, transient expression of a full-length Relish protein, modified by an N-terminal FLAG tag, was induced in the same S2 cells and the effects of the SkpA and slimb RNAi treatments on FLAG-Relish protein stability was monitored using Western blots and anti-FLAG antibodies (Khush, 2002).

Reducing Slimb activity, in the absence of LPS stimulation, visibly increases steady-state levels of both full-length Relish and the active N-terminal Rel-homology domain; levels of both polypeptides are further increased by inhibiting Slimb and SkpA simultaneously. This effect is specific since RNAi of the SkpA homologs, SkpB and SkpD, does not increase Relish levels. Dredd RNAi does increase Relish levels at day 1, but this is probably because Dredd inhibition blocks Relish processing. Previous studies show that Relish processing in S2 cells is induced by lipopolysaccharide (LPS) and requires Dredd activity. As expected, therefore, RNAi of Dredd blocks LPS-induced Relish processing. Simultaneous RNAi of SkpA and Slimb in the presence of LPS, however, results in higher steady-state levels of the Rel-homology domain up to 4 days after Relish induction. Higher levels of the Rel-homology domain after SkpA and Slimb RNAi could be caused by increased processing of full-length Relish. However, because full-length Relish levels also mount, the explanation is favored that Rel-homology domain turnover is reduced. Although the Slimb and SkpA RNAi treatments appear to inhibit Relish turnover, Relish levels do eventually diminish. This suggests that RNAi efficiency decreases with time, possibly due to degradation of the transfected double-stranded RNA. These RNAi experiments indicate that the constitutive antimicrobial gene expression in SkpA and slimb mutant flies is caused by higher Relish levels, and they suggest that the SCFSkpA/Cul1/Slimb complex represses the IMD pathway by promoting the degradation of both full-length and processed Relish proteins (Khush, 2002).

If the constitutive antimicrobial gene expression in flies carrying mutations that affect the SCFSkpA/Cul1/Slimb complex or proteasome is due to higher Relish levels, this would imply some level of steady-state Relish activation. Low levels of the Rel-homology domain have been reported in nuclear extracts from unstimulated S2 cells, and these low levels indicate that Relish is constitutively processed. Increasing Relish levels in larvae and adults via the Gal4-UAS system is sufficient to induce low levels of Diptericin expression. These results indicate that Relish is constitutively processed and activated to some level, supporting the hypothesis that Relish activity, in the absence of infection, is countered by ubiquitination and degradation (Khush, 2002).

Multiple roles of the F-box protein Slimb in Drosophila egg chamber development

Substrate-specific degradation of proteins by the ubiquitin-proteasome pathway is a precise mechanism that controls the abundance of key cell regulators. SCF complexes are a family of E3 ubiquitin ligases that target specific proteins for destruction at the 26S-proteasome. These complexes are composed of three constant polypeptides -- Skp1, Cullin1/3 and Roc1/Rbx1 -- and a fourth variable adapter, the F-box protein. Slimb (Slmb) is a Drosophila F-Box protein that fulfills several roles in development and cell physiology. Slmb participation in egg chamber development was analyzed and slmb was found to be required in both the follicle cells and the germline at different stages of oogenesis. In slmb somatic clones, morphogenesis of the germarium and encapsulation of the cyst are altered, giving rise to egg chambers with extra germline cells and two oocytes. Furthermore, in slmb somatic clones, ectopic Fasciclin 3 expression was observed, suggesting a delay in follicle cell differentiation, that correlates with the occurrence of ectopic polar cells, lack of interfollicular stalks and mislocalization of the oocyte. Later in oogenesis, Slmb is required in somatic cells to specify the position, size and morphology of dorsal appendages. Mild overactivation of the Dpp pathway causes similar phenotypes that are antagonized by simultaneous overexpression of Slmb, suggesting that Slmb normally downregulates the Dpp pathway in follicle cells. Indeed, ectopic expression of a dad-LacZ enhancer trap reveals that the Dpp pathway is upregulated in slmb somatic clones and, consistent with this, ectopic accumulation of the co-Smad protein, Medea, occurs. By analyzing slmb germline clones, it was found that loss of Slmb provokes a reduction in E2f2 and Dp levels, which correlate with misregulation of mitotic cycles during cyst formation, abnormal nurse cell endoreplication and impairment of dumping of the nurse cell content into the oocyte (Muzzopappa, 2005).

Thus Slmb is required for oogenesis in both the germline and FC. In the germline, Slmb plays a role in the control of mitotic cycles during cyst formation, in regulation of nurse cell endoreplication and in nurse cell dumping. Recent reports have demonstrated that Slmb can control cell cycle progression in different experimental settings. Following DNA replication, Slmb is required in larval wing discs for proteolysis of the cell cycle modulator E2f1. Remarkably, the E2f complex is implicated in cell cycle control of ovarian germ cells, in nurse cell transition from polyteny to polyploidy and in dumping of the nurse cell content into the oocyte. This study shows that two subunits of the E2f complex, Dp and E2f2, are downregulated in ovaries bearing slmb germline clones, while E2f1 does not change. Differences in Cyclin E levels, another cell cycle regulator involved in cyst formation, could not be detected in these clones. A good correlation exists between the phenotypes observed in slmb germline clones and in Dp germline clones; in both cases an additional round of cystocyte mitotic divisions occurs. In order to understand the molecular mechanism causing Dp and E2f2 reduction in slmb germline clones, a detailed analysis of the alterations of the network regulating the cell cycle is required (Muzzopappa, 2005).

Although expression levels in somatic cells in the germarium are too low to be detected through an enhancer trap or by in situ hybridization, loss-of-function experiments suggest that slmb is needed in these cells for normal morphogenesis of the egg chamber and for encapsulation of the cyst. In addition, the results suggest that Slmb is required for timely differentiation of FC that is reflected by the refinement of Fas3 expression; this is accompanied by the occurrence of ectopic polar cells, lack of interfollicular stalks and disruption of normal egg chamber polarity. Later in oogenesis, Slmb is expressed at high levels in FC surrounding the oocyte and participates in chorion patterning, contributing to define the shape and position of DA (Muzzopappa, 2005).

It has been reported that slmb mutant clones induce ectopic activation of the Hedgehog (Hh) pathway in limb discs. Notably, some of the phenotypes observed upon slmb somatic clone induction are similar to those originated by overactivation of the Hh pathway in FC. These include a delay in FC differentiation, development of ectopic polar cells and mislocalization of the oocyte. Nevertheless, excessive activation of the Hh pathway also causes FC over-proliferation that results in excess of undifferentiated somatic cells that form very long interfollicular stalks between egg chambers. By contrast, slmb loss of function in FC caused a lack rather than an excess of interfollicular cells. Finally, dominant genetic interactions were not detected between slmb and negative regulators of Hh pathway and the ptc-LacZ enhancer trap, which has been reported to be activated in FC by the Hh pathway, is not induced ectopically in slmb mutant clones. These results indicate that, despite some similarities between slmb loss-of-function and hh gain-of-function phenotypes, Slmb is unlikely to be a negative regulator of Hh pathway during oogenesis (Muzzopappa, 2005).

In limb discs, Slmb is a negative regulator of the Dpp pathway, although the molecular mechanism involved is still unclear. Mild overexpression of Dpp causes a wide spectrum of phenotypes that are largely coincident with those caused by slmb loss of function in FC. Supporting the idea that loss of slmb might cause hyperactivation of the Dpp pathway, the strongest chorion phenotypes originated by overexpression of Dpp are completely antagonized by simultaneous overexpression of Slmb in FC. Moreover, expansion of dad-lacZ expression occurs in slmb mutant follicles, further suggesting that ectopic induction of the Dpp pathway indeed occurs as a consequence of slmb loss of function. Consistent with this, a downstream component of the Dpp pathway, the co-Smad protein Medea, is upregulated in slmb mutant egg chambers. Because in mammalian cell culture it was demonstrated that Smad4 is a direct target of the mammalian Slmb ortholog, ßTrcp1, it is believed that Medea could be a direct target of Slmb. Further molecular work is required to assess whether this is indeed the case or if alternatively, the effect of Slmb on Medea is indirect (Muzzopappa, 2005).

Wnt signaling cross-talks with JH signaling by suppressing Met and gce expression

Juvenile hormone (JH) plays key roles in controlling insect growth and metamorphosis. However, relatively little is known about the JH signaling pathways. Until recent years, increasing evidence has suggested that JH modulates the action of 20-hydroxyecdysone (20E) by regulating expression of broad (br), a 20E early response gene, through Met/Gce and Kr-h1. To identify other genes involved in JH signaling, a novel Drosophila genetic screen was designed to isolate mutations that derepress JH-mediated br suppression at early larval stages. It was found that mutations in three Wnt signaling negative regulators in Drosophila, Axin (Axn), supernumerary limbs (slmb), and naked cuticle (nkd), caused precocious br expression, which could not be blocked by exogenous juvenile hormone analogs (JHA). A similar phenotype was observed when armadillo (arm), the mediator of Wnt signaling, was overexpressed. qRT-PCR revealed that Met, gce and Kr-h1expression are suppressed in the Axn, slmb and nkd mutants as well as in arm gain-of-function larvae. Furthermore, ectopic expression of gce restored Kr-h1 expression but not Met expression in the arm gain-of-function larvae. Taken together, it is concluded that Wnt signaling cross-talks with JH signaling by suppressing transcription of Met and gce, genes that encode for putative JH receptors. The reduced JH activity further induces down-regulation of Kr-h1expression and eventually derepresses br expression in the Drosophila early larval stages (Abdou, 2011).

JH transduces its signal through Methoprene-tolerant (Met), Germ cell-expressed (Gce) and Krüppel-homolog 1 (Kr-h1) and the p160/SRC/NCoA-like molecule (Taiman in Drosophila and FISC in Aedes). The Drosophila Met and gce genes encode two functionally redundant bHLH-PAS protein family members, which have been proposed to be components of the elusive JH receptor. Both Met and gce mutants are viable and resistant to JH analogs (JHA) as well as to natural JH III. However, Met-gce double mutants are prepupal lethal and phenocopies CA-ablation flies. The Met protein binds JH III with high affinity. In Tribolium, suppression of Met activity by injecting double-stranded (ds) Met RNA causes precocious metamorphosis. Kr-h1 is considered as a JH signaling component working downstream of Met. In both Drosophila and Tribolium, Kruppel-homolog1 (Kr-h1) mRNA exhibits high levels during the embryonic stage and is continuously expressed in the larvae; then, it disappears during pupal and adult development. Kr-h1 expression can be induced in the abdominal integument by exogenous JH analog (JHA) at pupariation. Suppression of Kr-h1 by dsRNA in the early larval instars of Tribolium causes precocious br expression and premature metamorphosis after one succeeding instar. Thus, Kr-h1 is necessary for JH to maintain the larval state during a molt by suppressing br expression. Studies in Aedes, Drosophila and Tribolium have demonstrated that the p160/SRC/NCoA-like molecule is also required for JH to induce expression of Kr-h1 and other JH response genes. For example, Aedes FISC forms a functional complex with Met on the JH response element in the presence of JH and directly activates transcription of JH target genes (Abdou, 2011 and references therein).

In an attempt to isolate other genes involving JH signaling, a novel genetic screen was conducted, and mutations in were identified in three Wnt signaling component genes, Axin (Axn), supernumerary limbs (slmb), and naked cuticle (nkd), induced precocious br expression, which was similar to a loss of JH activity. The evolutionarily conserved Wnt signaling pathway controls numerous developmental processes. The key mediator of the Drosophila Wnt pathway is Armadillo (Arm, the homolog of vertebrate β-catenin). When the Wnt signaling ligand, Wingless (Wg), is absent, the destruction complex is active and phosphorylates Arm, earmarking it for degradation. Upon Wg stimulation, the destruction complex is inactivated; as a result, unphosphorylated Arm accumulates in the cytosol and is targeted to the nucleus to stimulate transcription of Wnt target genes. Many players in the Wnt signaling pathway negatively regulate its activity. For example, Axin (Axn) is one of the main components of the destruction complex. Supernumerary limbs (Slmb) recognizes phosphorylated Arm and targets it for polyubiqitination and proteasomal destruction. Naked cuticle (Nkd) antagonizes Wnt signaling by inhibiting nuclear import of Arm. The current investigations reveal that the high activity of Wnt signaling in the Axn, slmb, and nkd mutants suppresses the transcription of Met and gce, genes encoding for putative JH receptors, thus linking Wnt signaling to JH signaling and insect metamorphosis for the first time (Abdou, 2011).

The 'status quo' action of JH in controlling insect metamorphosis is conserved in hemimetabous and most holometabous insects. However, the larval-pupal transition in higher Diptera, such as Drosophila, has largely lost its dependence on JH. For instance, in most insects, the addition of JH in larvae at the last instar causes the formation of supernumerary larvae. However, exogenous JH does not prevent pupariation and pupation in Drosophila, and instead disrupts the development of only the adult abdominal cuticle and some internal tissues. The molecular mechanisms underlying these differential responses to JH are not clear (Abdou, 2011).

Broad is a JH-dependent regulator that specifies pupal development and mediates the 'status quo' action of JH. In the relatively basal holometabolous insects, such as beetles and moths, JH is both necessary and sufficient to repress br expression during all of the larval stages. These studies revealed that JH is also required during the early larval stages in the more derived groups of the holometabolous insects, such as Drosophila, but it is not sufficient to repress br expression at the late 3rd instar. During the early larval stages, overexpression of the JH-degradative enzyme JHE, reduction of JH biosynthesis or disruption of the JH signaling always causes precocious br expression in the fat body. However, exogenous JHA treatment can not repress br expression in the fat body of late 3rd instar larvae. The molecular mechanism underlying the developmental stage-specific responses of the br gene to JH signaling remains to be clarified (Abdou, 2011).

As knowledge of signal transduction increases, the next step is to understand how individual signaling pathways integrate into the broader signaling networks that regulate fundamental biological processes. In vertebrates, Wnt signaling has been found to interact with different hormone signaling pathways to mediate various developmental events. For example, the Wnt/beta-catenin signaling pathway interacts with thyroid hormones in the terminal differentiation of growth plate chondrocytes and interacts with estrogen to regulate early gene expression in response to mechanical strain in osteoblastic cells. In insects, both Wnt and JH signaling are important regulatory pathways, each controlling a wide range of biological processes. This study reports that the Wnt signaling pathway interacts with JH in regulating insect development. During the Drosophila early larval stages, elevated Wnt signaling activity in the Axn, slmb, nkd mutants and arm-GAL4/UAS-armS10 flies represses Met and gce expression, which down-regulates Kr-h1 and causes precocious br expression in the fat body. Ectopic expression of UAS-gce in the arm-GAL4/UAS-armS10 larvae is sufficient for restoring Kr-h1 expression and then repressing br expression (Abdou, 2011).

Arm is a co-activator that interacts with Drosophila TCF homolog Pangolin (Pan), a Wnt-response element-binding protein, to stimulate expression of Wnt signaling target genes. In the absence of nuclear Arm, Pan interacts with Groucho, a co-repressor, to repress transcription of Wingless-responsive genes. Upon the presence of nuclear Arm, it binds to Pan, converting it into a transcriptional activator to promote the transcription of Wingless-responsive genes. It is proposed that Wnt signaling indirectly suppresses Met and gce expression by activating an unknown transcriptional repressor (Abdou, 2011).

JH signaling is well known to be a systemic factor that decides juvenile versus adult commitment. Wg is a morphogen that tissue-autonomously promotes proliferation and patterning during organogenesis. The current studies show that ectopically activating Wg signaling, either by mutations of negative regulators or by the ectopic expression of Arm, results in br derepression via loss of Met and Gce. How and why does the localized Wg signaling regulate the global JH signaling during insect development? It is hypothesized that though JH signaling activity is globally controlled by JH titer in the hemolymph, distinct tissues may response to JH with different sensitivity, which could be regulated by Wnt signaling-mediated Met and gce expression. Actually, it was found that precocious br expression is detectible in the fat body but not midgut of the Axn mutant 2nd instar larvae. This is one line of evidence to support that Wnt signaling regulates Met and gce expression in a tissue-specific manner (Abdou, 2011).


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supernumerary limbs: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 December 2014

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