Analysis of dSno transcription utilizing Northern blots identified two strongly hybridizing transcripts that correspond in size to dSnoN and dSnoN2 cDNAs in third instar larvae. One of these transcripts is weakly visible in adult females. A prominent smaller band, also visible in L3, does not correspond to any of the sequenced cDNAs but could represent an alternately spliced version of either SnoA or SnoI (Takaesu, 2006).
In embryos, dSno expression is not visible until embryonic stage 14. Thus, given the large number of functions for BMP signaling that precede stage 14, the absence of early expression suggests that dSno is not utilized as a universal terminator of BMP signaling, a possibility suggested by studies of Sno in mammals. At stage 14, dSno expression is widespread in the ectoderm with a slightly increased concentration in the ventral ectoderm. At stage 16, dSno transcripts are still widespread in the ectoderm but they are now most prominent in the brain and ventrally located nerve cord of the central nervous system. At stage 17, dSno expression is completely lost outside the CNS while CNS expression remains strong. Within the ventral nerve cord segmentally reiterated peaks of expression are visible (Takaesu, 2006).
As expected from the Northern, dSno is strongly expressed in third instar larval discs. dSno expression is widespread in leg discs with perhaps a slight increase in expression in a semicircular pattern corresponding to the most proximal regions. In contrast, dpp expression is localized along the anterior–posterior compartment boundary with expression in proximal and distal regions. dSno expression in the wing blade appears as a semicircular pattern that excludes distal regions. In contrast, dpp expression is localized along the anterior–posterior compartment boundary with prominent expression in the wing blade, including distal regions. dSno expression is widespread in the eye/antennal disc and there is a decrease in expression in the differentiating neurons behind the morphogenetic furrow. In contrast, dpp expression is localized along the morphogenetic furrow in the eye disc and in the ventral region of the antennal disc. The fact that the expression patterns for dpp and dSno are only partially overlapping raised the question of whether BMP antagonism was a function of endogenous dSno (Takaesu, 2006).
Signalling by TGF-β ligands through the Smad family of transcription factors is critical for developmental patterning and growth. Disruption of this pathway has been observed in various cancers. In vertebrates, members of the Ski/Sno protein family can act as negative regulators of TGF-β signalling, interfering with the Smad machinery to inhibit the transcriptional output of this pathway. In some contexts ski/sno genes function as tumour suppressors, but they were originally identified as oncogenes, whose expression is up-regulated in many tumours. These growth regulatory effects and the normal physiological functions of Ski/Sno proteins have been proposed to result from changes in TGF-β signalling. However, this model is controversial and may be over-simplified, because recent findings indicate that Ski/Sno proteins can affect other signalling pathways. To address this issue in an in vivo context, the function of the Drosophila Ski/Sno orthologue, SnoN was analyzed. SnoN was found to inhibit growth when overexpressed, indicating a tumour suppressor role in flies. It can act in multiple tissues to selectively and cell autonomously antagonise signalling by TGF-β ligands from both the BMP and Activin sub-families. By contrast, analysis of a snoN mutant indicates that the gene does not play a global role in TGF-β-mediated functions, but specifically inhibits TGF-β-induced wing vein formation. It is proposed that SnoN normally functions redundantly with other TGF-β pathway antagonists to finely adjust signalling levels, but that it can behave as an extremely potent inhibitor of TGF-β signalling when highly expressed, highlighting the significance of its deregulation in cancer cells (Ramel, 2006).
SnoN overexpression produced a range of different patterning and growth phenotypes in the eye, wing and other tissues. The patterning defects observed are consistent with reduced TGF-β signalling, e.g. loss of wing veins with wing GAL4 drivers and failure of thorax closure when expressed in presumptive thoracic body wall epithelium. Dpp and Activin signalling have also been implicated in driving growth in the wing and so the growth inhibitory activity of SnoN in this tissue also fits with its proposed antagonistic function. Growth regulation by TGF-β signalling in the eye has been less extensively studied, but it was possible to show that increasing either Dpp or Activin signalling primarily in differentiating cells promotes overgrowth and this is strongly suppressed by co-overexpression of SnoN. When SnoN is expressed by itself in the differentiating eye, its growth inhibitory effects appear to be entirely due to reduced cell growth and cell size. Even though the disorganized patterning seen in eyes with excess TGF-β signalling may result from changes in cell number as well as cell size, these effects are also potently suppressed by SnoN (Ramel, 2006).
It should be noted that Activin signalling was less efficiently suppressed by SnoN compared to Dpp signalling in both the eye and wing. Although the data support a role for SnoN in inhibiting Activin functions, the possibility that there is cross-talk between the two TGF-β pathways cannot be excluded, particularly in these overexpression assays, and that suppression of Activin’s effects on the Dpp target Smad Mad are being observed. Resolution of this issue will require the identification of a specific Activin target gene in the wing or eye, as has been used for the Dpp pathway (Ramel, 2006).
Overgrowth phenotypes are also observed upon overexpression of components of the InR signalling pathway. Interestingly, SnoN overexpression was unable to suppress the overgrowth generated by Akt1 overexpression, indicating that SnoN specifically acts downstream of TGF-β signalling. In fact, overexpressed Akt1 suppressed the growth inhibitory effects of SnoN. This is consistent with previous observations that the effects of Tkv overexpression on growth are at least partly mediated by components of the InR signalling cascade (Ramel, 2006).
By studying the regulation of an established Dpp target gene, omb, in the wing disc through clonal analysis, it was found that SnoN inhibits Dpp signalling cell autonomously. Not only does this support the hypothesis that SnoN directly modulates TGF-β signalling in flies, but the cell autonomous behaviour of this molecule is consistent with its proposed role as a transcriptional modulator (Ramel, 2006).
In vertebrates, Ski/Sno proteins have been implicated in the regulation of several different signalling cascades, including those involving Hedgehog and Wnt family proteins (Dai, 2002; Chen, 2003), but the relevance of these interactions in vivo has remained unclear. Overexpression data cannot exclude a role for SnoN in these other signalling pathways, but they do suggest a primary function in TGF-β signalling, a conclusion further supported by mutant analysis. Although the SnoN-TGF-β link has been suggested previously in vertebrate systems, primarily through overexpression approaches in cell culture, analysis in Drosophila confirms the importance of this process in vivo. In addition, the results are also consistent with the idea that SnoN’s effects on growth in whole animals may all be mediated through changes in TGF-β signalling, which in Drosophila primarily plays a growth-promoting role. Hence in flies, SnoN acts as a tumour suppressor, but just as in vertebrates, its effects on growth are most clearly observed when overexpressed (Ramel, 2006).
It was surprising to find that flies homozygous for the snoNGS-C517T mutant allele develop with only minor patterning defects (ectopic wing veins). This suggests that SnoN has a highly restricted role in development. Since in situ analysis indicates that snoN is expressed quite broadly in imaginal discs, this restricted role cannot be explained merely by localized gene expression (Ramel, 2006).
It is believed that snoNGS-C517T is likely to represent a strong loss-of-function allele for three reasons. (1) It is predicted to produce a protein that lacks the entire evolutionarily conserved SAND domain, which interacts with Smad4 (co-Smad), and about one third of the Ski/Sno family domain, so it should not be able to antagonize the TGF-β signalling pathway. It also lacks regions required in mammalian Ski for binding to Gli3 (Hedgehog pathway; Dai, 2002) and FHL2 (Wnt pathway; Chen, 2003). Thus, even if fly SnoN could interact with these signalling cascades, it is predicted that the mutant protein would not. (2) Overexpression phenotypes observed with the snoNGS18054 insertion are all completely reverted by the mutation, indicating that the allele has lost its biological activity. (3) Ubiquitous overexpression of the putative dominant negative snoNGS-C517T allele precisely phenocopies the homozygous mutant phenotype. Thus, the results suggest that Drosophila SnoN has no essential role in the majority of TGF-β-dependent events, despite its potent activity as a TGF-β signalling antagonist in flies and vertebrates. The developmental functions of snoN in mammalian development are not yet clear. Indeed, loss of SnoN function has been shown to be embryonic lethal (Shinagawa, 2000), but another group (Pearson-White, 2003) reported that two different snoN mouse mutants are viable and only show T-cell activation defects (Ramel, 2006).
Several lines of evidence confirm that the ectopic wing vein phenotype observed in snoNGS-C517T homozygous flies results from inhibition of a SnoN-dependent function. First and most importantly, expression of wild-type SnoN with bs1348-GAL4 largely suppresses the snoNGS-C517T ectopic wing vein phenotype. In addition, the similarity between the mutant phenotype and the defects observed in situations where TGF-β activity is up-regulated, as well as the genetic interaction data with dpp alleles are fully consistent with the ectopic wing vein phenotype resulting from loss of a TGF-β signalling antagonist, such as SnoN (Ramel, 2006).
The snoNGS-C517T allele also acts in a dominant negative fashion. Indeed, it specifically suppresses the normal function of SnoN (wing vein development inhibition), since strong constitutive overexpression of snoNGS-C517T accurately phenocopies the homozygous mutant phenotype. Moreover, in snoNGS-C517T, tub-GAL4 discs, ectopic P-Mad expression was observed specifically in the region around L5, where the adult phenotype is observed. This last result might appear to contradict models for vertebrate Ski/Sno function, in which these molecules act downstream of Smad activation. However, the pattern of P-Mad expression outside the longitudinal proveins is thought to evolve in a complex process involving long-range signalling and feedback regulation. Thus, increased Dpp transcriptional output in a snoN mutant could subsequently lead to increased P-Mad expression (Ramel, 2006).
Takaesu (2006) suggests that the lethal mutation in the l(2)SH1402 chromosome is caused by loss of the 297{}323 retrotransposon affecting snoN expression, but this transposon was found to be absent even in wild-type flies. Thus, another as yet unidentified mutation must be responsible for the lethality. Surprisingly, it was found that l(2)SH1402 complements the snoN deficiency chromosome used in the current study. The boundaries of this deficiency were confirmed and the absence of the 5′ end of the snoN gene was specifically shown in the deficiency by PCR. l(2)SH1402 also complements the snoNGS-C517T mutant, because l(2)SH1402/snoNGS-C517T animals are viable and display an ectopic wing vein phenotype at a frequency similar to snoNGS-C517T heterozygous flies. These results suggest that the lethality observed in l(2)SH1402 flies is not entirely due to a disruption of SnoN’s function, despite the fact that constitutive overexpression of snoN is reported to rescue the lethal phenotype. In light of these observations, it is therefore believed that the snoNGS-C517T allele has retained little if any normal function and that the homozygous snoNGS-C517T phenotype, therefore, reflects the fact that this gene is not essential for viability in vivo (Ramel, 2006).
One inconsistency in the data is that ectopic wing vein frequency is reduced in snoNGS-C517T /Df(2L)ED12527 flies relative to homozygous snoNGS-C517T animals, suggesting that the effects of snoNGS-C517T are more severe than a complete loss-of-function allele. Evidence is provided that there may be another mutation in the deficiency region that represses TGF-β signalling, since this chromosome also partially suppresses the dominant negative effects of overexpressed SnoNGS-C517T. However, the possibility cannot be eliminated that part of the snoNGS-C517T homozygous mutant phenotype is caused by a dominant effect on other regulators of the TGF-β signalling cascade and this could explain the fact that overexpressed SnoN does not fully rescue this phenotype (Ramel, 2006).
In this regard, it is already known that, in the process of pupal wing vein formation, at least three antagonists of Dpp signalling activity, Short-gastrulation (Sog), Brinker, and Daughter-against-Decapentaplegic, are able to block wing vein development upon overexpression. The intervein expression pattern of SnoN in pupal wings is similar to the expression of Sog, an extracellular molecule that inhibits Dpp ligand binding, and Brinker, which acts as a transcriptional repressor of Dpp target genes. Analysis of sog mutant clones in the wing suggests that Sog is required to limit longitudinal vein formation to the provein regions. The wings of brinker mutant escapers display ectopic wing vein tissue, as do brinker mutant clones. If the dominant negative SnoNGS-C517T protein can still complex to some of the molecules involved in Dpp signalling without inhibiting them, this might partially block the ability of these alternative antagonists to compensate for loss of normal SnoN function and therefore, produce a phenotype more severe than a snoN null allele (Ramel, 2006).
In conclusion, a model is proposed in which Drosophila SnoN normally plays a highly restricted role in TGF-β-dependent events. During development, the sensitivity of TGF-β signalling to SnoN levels may be important in providing a responsive mechanism to fine-tune and balance fluctuations in signalling. One prediction of this model is that snoN mutant phenotypes would be highly sensitive to the genetic background and to levels of TGF-β signalling, both of which were observed. The use of multiple potent, but partially redundant, inhibitors to control a fundamental signalling cascade is a powerful mechanism for maintaining stable levels of signalling activity. However, for snoN, such a mechanism carries the inherent risk that it can cause severe defects if its expression is altered, as is frequently observed in tumours (Ramel, 2006).
The pronounced expression of dSno in the CNS beginning at embryonic stage 16 prompted an examination of this tissue for developmental defects in dSno mutants. BMPs regulate the growth of the neuromuscular junction synapse and phosphorylated Mad (pMad) normally accumulates in motoneuron nuclei beginning at embryonic stage 15. However, no alteration was seen in the intensity or pattern of pMad accumulation in the CNS, or in any other embryonic tissue, in dSno mutant embryos. The neuromuscular junction of dSno mutant third instar larvae was examined with the presynaptic marker Csp and no obvious difference was observed in bouton numbers or overall synapse size. Together, these results suggest that dSno does not modulate BMP signaling in motor neurons (Takaesu, 2006).
The fact that the majority of dSno mutants die after pupation without differentiating into pharate adults is similar to observations of strong baboon (babo) and dSmad2 mutants. Baboon is an Activin type I receptor and babo and dSmad2 phenotypes are being characterized in more detail. In these mutants pronounced optic lobe defects were observed related to photoreceptor innervation of the lamina and medulla. Axons of photoreceptors R1–6 normally terminate at the lamina, while R7–8 project deeper into the brain, forming an elaborate lattice-like network with expanded growth cones that make contact with the medulla neuropil. In both babo and dSmad2 mutants, R1–6 axons project into the brain and form a relatively normal but very reduced lamina plexus while R7 and R8 axons never form a normal lattice network and their growth cones are collapsed. Intriguingly, dSno mutant larvae exhibit a very similar phenotype (Takaesu, 2006).
It was also found that the medulla neuropil is significantly reduced in size and its morphology is altered in babo and dSmad2 mutants. Again, dSno mutant larval brains show a similar alteration in medulla neuropil size and architecture. In both babo and dSmad2 mutants, these defects likely result as a secondary consequence of reduced cell proliferation within the optic lobes of the brain. Likewise, it was found that dSno mutants show reduced numbers of cells in M phase as assayed by phospho-histone H3 staining (Takaesu, 2006).
These results are consistent with the hypothesis that dSno mediates Babo signaling in the optic lobes to maintain correct proliferation rates in neuroblasts and/or their daughter cells during larval development. However, no direct transcriptional targets of Babo signaling that regulate proliferation in the optic lobe are known. At present, only the ecdysone receptor-1B isoform (EcR-1B) has been identified as a target of Babo and dSmad2 signaling in mature neurons of the CNS. In the absence of either gene, EcR-1B expression is dramatically reduced. In contrast, dSno mutants exhibit apparently normal levels of EcR-1B expression. This result implies that dSno is not required for Babo-mediated responses in mature neurons of the CNS (Takaesu, 2006).
dSno loss-of-function mutations were analyzed to address the possibility that BMP antagonism is a function of engogenous dSno. A strain bearing the l(2)sh1402 chromosome was used. This chromosome is lethal when homozygous and it was verified by Southern blot that it contains a single P-element insertion. Plasmid rescue verified that the P element is located 735 bp upstream of CG7233 (dSno). Before commencing a detailed analysis of l(2)sh1402, the lethality of P-element insertion was examined and whether the P-element insertion affects dSno activity (Takaesu, 2006).
To ensure that the P-element insertion was associated with the lethality of the l(2)sh1402 chromosome, a recombination mapping experiment was conducted. If the mini-white marked P element was responsible for the lethality, then it was predicted that, after allowing the l(2)sh1402 chromosome to freely recombine with a wild -type chromosome, the descendant mini-white-marked chromosomes would be homozygous lethal and non-mini-white chromosomes would be homozygous viable. More than 90 recombinant chromosomes of each type was sibmated and least 50 progeny from each sibmate were scored. Consistent with predictions, it was found that mini-white chromosomes were lethal and that non-mini-white chromosomes were viable (Takaesu, 2006).
To ensure that the P-element insertion in l(2)sh1402 affected only dSno function, complementation tests were conducted with three P-element insertion chromosomes associated with the adjacent CG7231 (upstream of dSno). P{wHy}CG7231 contains an insertion in the 5' untranslated region of the first protein coding exon of CG7231 on the basis of EST data. This chromosome is homozygous lethal but there is a moderate percentage of escapers. P{SUPor-P}CG7231[KG04307]) has an insertion just upstream of the 5' untranslated region of CG7231 and it is also homozygous lethal with escapers. P{EPgy2}EY11884 has an insertion in an intron of CG7231 and it is fully viable and fertile as a homozygote. The l(2)sh1402 chromosome fully complemented each of these chromosomes, indicating that its P-element insertion does not affect CG7231 function. l(2)sh1402 is hereafter referred to as dSnosh1402 (Takaesu, 2006).
Surprisingly, the lethal dSnosh1402 chromosome fully complemented all known deletions affecting the 28D-E region. Of these, complementation with Df(2L)BSC41 (28A4-B1; 28D3-9) was unexpected. The region where dSno is located (28D3,4) is supposedly deleted in this line. It was also found that dSnosh1402/Df(2L)BSC41 females were fully fertile. Therefore 200 excision chromosomes were generated from dSnosh1402 as evidenced by phenotypic loss of the mini-white marker carried on the transposon. All of these chromosomes were homozygous lethal and 100% lethal over the parental dSnosh1402 chromosome. This suggested that no precise excisions occurred, an unlikely scenario. In additional complementation tests, 195 of the 200 excision lines were fully viable and fertile over the all 28D-E deletion chromosomes. In the same tests, five excision lines showed varying levels of lethality with Df(2L)BSC41. These five lines fell into two classes. In class I (dSnoEX4B and dSnoEX17B), the number of viable progeny bearing the excision chromosome and Df(2L)BSC41 was ~40% of the expected number and these females were absolutely sterile. In class II (dSnoEX95A, dSnoEX34A, and dSnoEX71B), the number of progeny with the excision and Df(2L)BSC41 was ~60% of expected and these females were either weakly or fully fertile (Takaesu, 2006).
To clarify these complex complementation results, the stage of lethality for dSnosh1402 and the class I excision lines (dSnoEX4B and dSnoEX17B) was examined as homozygotes and as heterozygotes with dSnosh1402. dSnosh1402 homozygotes and dSnosh1402/dSnoEX17B individuals die at midpupal stage. Lethality for other genotypes occurred earlier: from first instar larval for dSnoEX4B/dSnoEX4B to the larval–pupal boundary for dSnosh1402/dSnoEX4B. dSnosh1402/dSnoEX4B animals pupariate but do not differentiate adult tissue. The nonembryonic lethality for all genotypes is consistent with late-stage dSno expression in embryos (Takaesu, 2006).
It was then asked if the lethality in these genotypes was specifically due to loss of dSno function in rescue experiments. Since constitutive ectodermal expression of UAS.dSno (e.g., with 32B.Gal4) is lethal, Hsp70.Gal4 was utilized to express UAS.dSno in each mutant. A variety of heat-shock regimes were used guided by the dSno temporal expression pattern and the stage of lethality for each genotype. It was found that application of a heat shock at 10 hr and again at 4 days of age was most successful. It was possible to rescue all of these absolutely lethal genotypes to adulthood except dSnoEX4B homozygotes (Takaesu, 2006).
Rescue was most robust for the dSnosh1402/dSnoEX17B trans-heterozygous genotype. Individuals of both sexes were recovered and ~53% of the expected adults were obtained. For the dSnoEX17B homozygous genotype, adults of both sexes were also recovered but at a slightly lower rate. Rescued adults of both genotypes had a held-out wing phenotype similar to that seen in dppd-ho mutants. This suggested that there was a delicate balance between providing enough dSno to rescue but not enough to antagonize Dpp signaling (Takaesu, 2006).
For three of the rescued genotypes (dSnosh1402/dSnosh1402, dSnoEX4B/dSnoEX17B, and dSnosh1402/dSnoEX4B), the number of recovered adults was 5–10% of expected and only males were obtained. It is speculated that in the ovary where abberant Dpp signaling can lead to the formation of germline stem cell tumors dSno function is exquisitely regulated and a fine-enough balance was not achieved. Alternatively, the lack of females may simply be stochastic, given the percentage of rescued adults. Overall, the rescue data suggest that the postembryonic lethality in all dSno mutant genotypes except dSnoEX4B homozygotes is due to the loss of dSno function (Takaesu, 2006).
The lesions of the dSnosh1402, dSnoEX4B, and dSnoEX17B chromosomes were characterized. The location of a P{lacW} insertion in dSnosh1402 was previously reported but only the 5'-end of the P was analyzed. Sequences flanking the 5'- and 3'-ends of the P element were analyzed and it was determined that the P was inserted precisely between nucleotides 155,798 and 155,799 of the genomic scaffold. The P element is in the 5' untranslated region of the exon containing the dSno initiator methionine in three of the four cDNAs that begin at the potential promoter 1 and four of the five cDNAs that begin at the proposed promoter 3 (Takaesu, 2006).
Unexpectedly, analysis of sequences flanking the 3'-end of the P element revealed a precise deletion of the 297 retrotransposon in dSnosh1402. The deletion begins 207 bp upstream of the P element and removes nucleotides 162,922–156,006 of the scaffold (6917 bp). The deletion completely removes the proposed promoter 2 and is predicted to impact transcription from the other putative promoters (promoter 1 and promoter 3). Nevertheless, in dSnosh1402 homozygotes, dSno embryonic expression appears wild type—consistent with survival to midpupal stage (Takaesu, 2006).
The deletion of the 297TE in dSnosh1402 suggests an explanation of why no homozygous viable excision lines were obtained. The 195 homozygous lethal excision lines that are also lethal over dSnosh1402, but viable and fully fertile over Df(2L)BSC41, contain precise excisions. The lethality in these lines may be due to homozygosity for the 297TE deletion. This suggests that a putative dSno promoter (promoter 2) that lies within the 297TE is required for viability and that the other five excision lines are probably imprecise excisions (Takaesu, 2006).
Molecular analysis shows that dSnoEX4B contains a deletion of >17.8 kb. The deletion removes the dSno homology domain at the 5'-end of all dSno proteins. Two of the proposed dSno promoters (promoter 2 and promoter 3) are also deleted. Mapping data on the proximal breakpoint were ambiguous and the presence of the putative promoter 1 is uncertain. However, the inability to rescue the lethality of this mutant with UAS.dSno suggests that one or more genes upstream of dSno are affected. Clearly, dSnoEX4B is a protein null allele (Takaesu, 2006).
dSnoEX17B contains a deletion of 1.5 kb. The distal breakpoint falls within the dSno homology domain while the proximal breakpoint does not extend beyond the 297TE deletion. The deletion truncates the 5'-end of all dSno proteins and two of the putative dSno promoters (promoter 2 and promoter 3) may be deleted. However, it is possible that a transcript from the potential promoter 1 could splice to a cryptic site downstream within the remaining open reading frame. Such a transcript could create a truncated protein initiated at methionine136 that would contain the region of the dSno homology domain containing the Smad4 interaction sites. Thus, dSnoEX17B is either a genetic null or a very strong hypomorph (Takaesu, 2006).
The complex complementation pattern of dSnosh1402 and its excision lines with Df(2L)BSC41 remained unexplained so the lesion in the Df(2L)BSC41 chromosome was characterized. Df(2L)BSC41 is a cytologically visible deletion removing sequences between 28A4-B1 and 28D3-9 that was generated by a dual P-element mobilization scheme. The distal element is P{lacW}l(2)k05404 in 28C7-9 and the proximal element is EP(2)0946 in 28D3-5. First, EP(2)0946 and transpose were placed in the same fly and then P{lacW}l(2)k05404 was added. In the third generation, according to the model, an interaction between the simultaneously mobilized P elements deletes all genetic material between them (Takaesu, 2006).
EP(2)0946 is located ~10 kb (scaffold 173,354) proximal to the most proximal exon of dSno. As a result, the model predicts that dSno (scaffold 163,026 - 71,178) is fully deleted in Df(2L)BSC41. However, when the Df(2L)BSC41 breakpoint in 28D3-9 was mapped, it was found to begin between scaffold 149,154 and 141,676 at a location ~30 kb proximal to the EP(2)0946 insertion point. The Df(2L)BSC41 deletion begins just upstream of the dSnoA unique exon and removes the 3'-end of the dSno locus. The 3'-ends of dSnoA, dSnoN, and dSnoN2 are absent, yet the three potential dSno promoters and the exon encoding the dSno homology domain are present. The shortest isoform (dSnoI) is present in its entirety (Takaesu, 2006).
A simple explanation for the discrepancy between predicted and actual breakpoints in Df(2L)BSC41 is that there was a local jump by EP(2)0946 in the first generation of the scheme. Then, after the second P element was added, the deletion producing interaction occurred. This possibility is supported by inverse PCR experiments indicating that in Df(2L)BSC41 neither P element remains in its original position, neither P element is intact, and both P elements made jumps during the mobilization scheme (Takaesu, 2006).
The observation that the deletion in Df(2L)BSC41 is the mirror image of the deletions in dSnoEX4B and dSnoEX17B provides an explanation for the reduction in fertility for these heterozygous females. dSnoI proteins generated from Df(2L)BSC41 are insufficient to perform all functions necessary for female fertility in the complete absence of dSno expression from dSnoEX4B and dSnoEX17B. The Df(2L)BSC41 breakpoint observation also provides an explanation for the fact that dSnosh1402 and its 195 precise excision lies are fully viable and fertile over this chromosome. In these heterozygous genotypes, dSnoI proteins expressed from Df(2L)BSC41 fully compensate for the absence of the proposed dSno promoter 2 in the precise excisions and for the additional effects of the P{lacW} insertion in dSnosh1402. Taken together, the analyses of Df(2L)BSC41 indicate that dSnoI can fulfill all dSno functions necessary for viability but not for female fertility (Takaesu, 2006).
The Drosophila eggshell is patterned by the combined action of the epidermal growth factor [EGF; Gurken (Grk)] and transforming growth factor ß [TGF-ß; Decapentaplegic (Dpp)] signaling cascades. Although Grk signaling alone can induce asymmetric gene expression within the follicular epithelium, the ability of Grk to induce dorsoventral polarity within the eggshell strictly depends on Dpp. Dpp, however, specifies at least one anterior region of the eggshell in the absence of Grk. Dpp forms an anteriorposterior morphogen gradient within the follicular epithelium and synergizes with the dorsoventral gradient of Grk signaling. High levels of Grk and Dpp signaling induce the operculum, whereas lower levels of both pathways induce the dorsal appendages (DAs). Evidence is presented that the crosstalk between both pathways occurs at least at two levels. First, Dpp appears to directly enhance the levels of EGF pathway activity within the follicular epithelium. Second, Dpp and EGF signaling collaborate in controlling the expression of Dpp inhibitors. One of these inhibitors is Drosophila sno (dSno), a homolog of the Ski/Sno family of vertebrate proto-oncogenes, which synergizes with daughters against dpp and brinker to set the posterior and lateral limits of the region, giving rise to dorsal follicle cells (Shravage, 2007).
The results show that Dpp has Grk-independent and Grk-dependent functions in the follicular epithelium. Even in the absence of Grk, Dpp is required to specify a group of anterior follicle cells that surround the micropyle. All dorsal follicle cells that contribute to a morphologically visible polarization of the eggshell require the combined action of Grk and Dpp. Within the region giving rise to dorsal follicle cells, Dpp acts together with Grk in a concentration-dependent manner to specify the identity and position of at least two distinct follicle cell types (Shravage, 2007).
In the absence of Dpp, Grk can still activate kekkon and repress pipe. Thus, Dpp is not required for Grk signaling per se. It is suggested that Dpp signaling rather activates transcription factors or causes chromatin modifications that allow Grk to induce dorsal target genes involved in follicle cell specification (Shravage, 2007).
Mirror might be such a transcription factor that is activated by Dpp and confers the ability to adopt dorsal fates to a ring of anterior follicle cells. mirror acts downstream of Grk and probably also downstream of Dpp in specifying dorsal follicle cells. However, mirror expression alone leads only to the formation of DA material. Thus, it is likely that mirror only provides the general potential to produce dorsal follicle cells. Additional inputs from Dpp and EGF signaling are needed to produce the full set of dorsal follicle cell fates. This scenario suggests two phases of Dpp signaling. An early phase demarcates the region in which Grk induces dorsal follicle cell fates. This might require only one (low level) threshold of Dpp signaling and is likely to be mediated through activation of mirror. A later phase establishes distinct dorsal follicle cell fates. Here, Dpp acts as a morphogen in combination with EGF signaling (Shravage, 2007).
The results presented in this study suggest that high levels of EGF and Dpp signaling correspond to regions II and III of the operculum, whereas lower levels of both pathways correspond to the DAs. With regard to region III of the operculum that separates the two DAs, the assumption appears to contradict a model based on results that showed that Grk signaling induces the expression of rhomboid (rho), which in turn activates Spitz, a second TGF-α-like molecule. This leads to an amplification of EGF signaling. Highest signaling levels centered at the dorsal midline lead to the induction of the inhibitor argos (aos), which antagonizes Spitz. This in turn lowers the levels of EGF signaling along the dorsal mildline. According to this model, high levels of EGF signaling promote DA, lower levels operculum region III formation. However, the expression patterns of kek, which result from Grk or Dpp overexpression, appear to contradict this model. Indeed, it is believed that the regulatory loop of rho and aos is not required to establish the operculum or DA fates per se. The pattern of BR-C expression is not significantly altered in rho or aos mutant follicle cell clones. However, rho and aos might contribute to patterning processes that are required for the morphogenesis and, as a result of this, for splitting of the DAs. DA extension (tube formation) has been shown to require the collaboration of rho-expressing floor cells and BR-C-expressing roof cells. The rho-expressing floor cells are part of the Fas3 expression domain that separates the BR-C domains. These rho-expressing cells have to form a separate stripe on each side of the dorsal midline to allow the splitting of the DAs. It is suggested that the rho/aos regulatory loop is required to generate two distinct stripes of late-rho expression within the dorsal Fas3 domain. The result is a splitting of the DAs accompanied by the establishment of a region of Fas3 cells that do not express rho, and thus give rise to region III of the operculum (Shravage, 2007).
The establishment of the region giving rise to dorsal follicle cells and its subdivision into operculum and DA-producing cells is an intriguing problem of two-dimensional patterning. The pattern of cell fates depends on the concentration-dependent read-out of two orthogonal signaling gradients (EGF and Dpp). This read-out is complex because the signaling pathways themselves appear to influence each other. (1) There is evidence for a direct influence of Dpp on EGF signaling; (2) the Dpp inhibitors brk and dSno are targets of both pathways, and (3) rho is also a target of both pathways (Shravage, 2007).
Evidence for a direct crosstalk between both pathways is provided by the analysis of kek expression. kek appears to be a primary target gene of EGF signaling, since basal levels of its expression are independent of Dpp. However, an enhancement of kek expression was observed upon dpp overexpression in stage 10A prior to the activation of rho and aos. Moreover, the stage 10B expression patterns of rho and aos do not correlate with the observed changes in kek expression. Thus, these changes cannot be caused by the secondary modulation of the EGF signaling profile. Therefore, a direct crosstalk between both pathways is suggested. This could be because of a Dpp receptor-dependent activation of the ras/MAPK cascade. A TGF-ß receptor-dependent activation of the MAPK cascade has been observed in several vertebrate cell types. One could imagine that the triangular-shaped domain of Fas3 expression, which defines the anterior and dorsal borders of the BR-C domain, is specified by high levels of EGF signaling brought about by a Dpp-dependent enhancement of MAPK signaling. A confirmation of this model would necessitate direct monitoring of MAPK activity upon altered Dpp signaling (Shravage, 2007).
The border between operculum and DAs is also crucially dependent on brk. In brk mutant follicle cell clones, Fas3 expression expands at the expense of the BR-C domains. However, brk expression is upregulated within a broad domain at the dorsal side that also includes the Fas3-expressing region separating the BR-C domains. Although brk represses Fas3 expression in lateral regions allowing BR-C expression, brk is unable to repress Fas3 at the dorsal midline. This suggests that Fas3 expression, which is predominantly dependent on high levels of EGF signaling, cannot be repressed by brk, whereas Fas3 expression in more lateral regions predominantly dependent on Dpp signaling is repressed by brk (Shravage, 2007).
The hemi-circular boundary of the total region giving rise to dorsal chorion fates appears to be defined by a constant value reflecting the sum or the product of EGF and Dpp signaling. The cis-regulatory elements of dSno represent a sensitive sensor for this dual input. At the dorsal midline, lower amounts of Dpp signaling are required to activate dSno than in lateral regions, and the opposite holds true for EGF signaling. During brain development in flies and in several contexts in vertebrates Sno is involved in the control of cell proliferation that has been shown to be crucially dependent on the relative levels of TGF-ß and EGF signaling. It is conceivable that for spatial patterning of the follicular epithelium dSno uses regulatory elements that are derived from a more basic function in the control of cell proliferation in other tissues. The follicle cell expression of dSno might provide a convenient experimental setting to dissect such regulatory elements (Shravage, 2007).
The fact that loss of dSno causes only mild defects is because of redundancy. A combination of three Dpp inhibitors appears to be involved in establishing the border between dorsal follicle cells and the remainder of the mainbody follicular epithelium. brk clones alone have no effect on the position of this border because they cause only a replacement of the DAs by operculum. dad mutant clones seem to lack patterning defects altogether. However, already removing one copy of these inhibitors in a homozygous dSno mutant background leads to an enlargement of operculum and a posterior shift of the DAs. Weak phenotypic effects of dSno have recently been reported for wing vein formation. Wing vein formation, too, represents a developmental context in which several Dpp inhibitors collaborate (Shravage, 2007).
The dSno mutation that was generated deletes a highly conserved protein domain that is responsible for the interaction with Smad proteins in vertebrates and with Medea in flies. The knockout mutations in mice are based on the deletion of this domain. Thus, this dSno mutation should represent a null allele. However, an unusual complexity of the dSno locus has been reported and a deletion is described that suggests that dSno is lethal, in variance to other findings. However, a truncation allele has been described lacking an important part of the conserved Smad interaction domain that, like the currently described allele, is viable. Because the possibility exists that the previously described deletion affects other genes in the chromosomal region of dSno, the question of lethality of dSno requires further analysis (Shravage, 2007).
Loss of dSno in the follicular epithelium does not result in changes in dpp expression or pMAD distribution. Whereas a feedback on dpp expression was not expected, possible changes in pMAD distribution might be below the level of detection of staining protocol. However, there are two other possible explanations. First, in brain development dSno has been shown to be a mediator of Baboon (Activin), rather than Dpp signaling. To investigate whether this also holds true for the follicular epithelium large baboon (Activin type I receptor) mutant follicle cell clones were generated. These clones did not show patterning defects, suggesting that dSno does not act via Baboon signaling with regard to follicle cell patterning. Second, the failure to detect changes in pMAD distribution might follow from the molecular mechanism of Sno action. A core feature of the inhibitory function of Sno proteins results from their ability to bind to the common Smad (Smad4). This binding prevents (or modulates) the interaction with phosphorylated R-Smads required for the transcriptional control of target genes. If this mechanism applies to DSno, the loss of dSno would not change the phosphorylation state of MAD and, if the interaction between DSno and Medea occurred predominantly in the nucleus, there would also be no significant change in the nuclear accumulation of pMAD (Shravage, 2007).
Signalling by TGF-β ligands through the Smad family of transcription factors is critical for developmental patterning and growth. Disruption of this pathway has been observed in various cancers. In vertebrates, members of the Ski/Sno protein family can act as negative regulators of TGF-β signalling, interfering with the Smad machinery to inhibit the transcriptional output of this pathway. In some contexts ski/sno genes function as tumour suppressors, but they were originally identified as oncogenes, whose expression is up-regulated in many tumours. These growth regulatory effects and the normal physiological functions of Ski/Sno proteins have been proposed to result from changes in TGF-β signalling. However, this model is controversial and may be over-simplified, because recent findings indicate that Ski/Sno proteins can affect other signalling pathways. To address this issue in an in vivo context, the function of the Drosophila Ski/Sno orthologue, SnoN, was analyzed. SnoN was found to inhibit growth when overexpressed, indicating a tumour suppressor role in flies. It can act in multiple tissues to selectively and cell autonomously antagonise signalling by TGF-β ligands from both the BMP and Activin sub-families. By contrast, analysis of a snoN mutant indicates that the gene does not play a global role in TGF-β-mediated functions, but specifically inhibits TGF-β-induced wing vein formation. It is proposed that SnoN normally functions redundantly with other TGF-β pathway antagonists to finely adjust signalling levels, but that it can behave as an extremely potent inhibitor of TGF-β signalling when highly expressed, highlighting the significance of its deregulation in cancer cells (Ramel, 2007).
SnoN overexpression produced a range of different patterning and growth phenotypes in the eye, wing and other tissues. The patterning defects observed are consistent with reduced TGF-β signalling, e.g. loss of wing veins with wing GAL4 drivers and failure of thorax closure when expressed in presumptive thoracic body wall epithelium. Dpp and Activin signalling have also been implicated in driving growth in the wing and so the growth inhibitory activity of SnoN in this tissue also fits with its proposed antagonistic function. Growth regulation by TGF-β signalling in the eye has been less extensively studied, but it was not possible to show that increasing either Dpp or Activin signalling primarily in differentiating cells promotes overgrowth and this is strongly suppressed by co-overexpression of SnoN. When SnoN is expressed by itself in the differentiating eye, its growth inhibitory effects appear to be entirely due to reduced cell growth and cell size. Even though the disorganized patterning seen in eyes with excess TGF-β signalling may result from changes in cell number as well as cell size, these effects were also potently suppressed by SnoN (Ramel, 2007).
It should be noted that Activin signalling was less efficiently suppressed by SnoN compared to Dpp signalling in both the eye and wing. Although the data support a role for SnoN in inhibiting Activin functions, the possibility that there is cross-talk between the two TGF-β pathways, particularly in these overexpression assays, cannot be excluded and that suppression of Activin's effects on the Dpp target Smad Mad is being observed. Resolution of this issue will require the identification of a specific Activin target gene in the wing or eye, as this study used for the Dpp pathway (Ramel, 2007).
Overgrowth phenotypes are also observed upon overexpression of components of the InR signalling pathway. Interestingly, it was found that SnoN overexpression was unable to suppress the overgrowth generated by Akt1 overexpression, indicating that SnoN specifically acts downstream of TGF-β signalling. In fact, overexpressed Akt1 suppressed the growth inhibitory effects of SnoN. This is consistent with observations that the effects of Tkv overexpression on growth are at least partly mediated by components of the InR signalling cascade (Ramel, 2007).
By studying the regulation of an established Dpp target gene, omb, in the wing disc through clonal analysis, it was found that SnoN inhibits Dpp signalling cell autonomously. Not only does this support the hypothesis that SnoN directly modulates TGF-β signalling in flies, but the cell autonomous behaviour of this molecule is consistent with its proposed role as a transcriptional modulator (Ramel, 2007).
In vertebrates, Ski/Sno proteins have been implicated in the regulation of several different signalling cascades, including those involving Hedgehog and Wnt family proteins, but the relevance of these interactions in vivo has remained unclear. The overexpression data cannot exclude a role for SnoN in these other signalling pathways, but they do suggest a primary function in TGF-β signalling, a conclusion further supported by the mutant analysis. Although the SnoN-TGF-β link has been suggested previously in vertebrate systems, primarily through overexpression approaches in cell culture, analysis in Drosophila confirms the importance of this process in vivo. In addition, the results are also consistent with the idea that SnoN's effects on growth in whole animals may all be mediated through changes in TGF-β signalling, which in Drosophila primarily plays a growth-promoting role. Hence in flies, SnoN acts as a tumour suppressor, but just as in vertebrates, its effects on growth are most clearly observed when overexpressed (Ramel, 2007).
Reference names in red indicate recommended papers.
Search PubMed for articles about Drosophila SnoN
Akiyoshi, S., et al. (1999). c-Ski acts as a transcriptional co-repressor in transforming growth factor-beta signaling through interaction with smads. J. Biol. Chem. 274(49): 35269-77. Medline abstract: 10575014
Arndt, S., Poser, I., Moser, M. and Bosserhoff, A. K. (2007). Fussel-15, a novel Ski/Sno homolog protein, antagonizes BMP signaling. Mol. Cell. Neurosci. [Epub ahead of print]. Medline abstract: 17292623
Bonaguidi, et al. (2005). LIF and BMP signaling generate separate and discrete types of GFAP-expressing cells. Development 132: 5503-5514. Medline abstract: 16314487
Briones-Orta, M. A., et al. (2006). SnoN co-repressor binds and represses smad7 gene promoter. Biochem. Biophys. Res. Commun. 341(3): 889-94. Medline abstract: 16442497
Chen, D., et al. (2003). Ski activates Wnt/β-catenin signalling in human melanoma. Cancer Res. 63: 6626-6634. Medline abstract: 14583455
Chen, W., et al. (2007). Competition between Ski and CBP for binding to Smads in TGF-β signaling. J. Biol. Chem. 282(15): 11365-76. Medline abstract: 17283070
da Graca, L. et al. (2004). DAF-5 is a Ski oncoprotein homolog that functions in a TGFβ pathway to regulate C. elegans dauer development. Development 131: 435-446. Medline abstract: 14681186
Dai, P., et al. (2002). Ski is involved in transcriptional regulation by the repressor and full-length forms of Gli3. Genes Dev. 16: 2843-2848. Medline abstract: 12435627
Egger, B., et al. (2002). Gliogenesis in Drosophila, analysis of downstream genes of glial cells missing in the embryonic nervous system. Development 129: 3295-3309. Medline abstract: 12091301
Hsu, Y. H., et al. (2006). Sumoylated SnoN represses transcription in a promoter-specific manner. J. Biol. Chem. 281(44): 33008-18. Medline abstract: 16966324
Kajino, T., Omori, E., Ishii, S., Matsumoto, K. and Ninomiya-Tsuji, J. (2007). TAK1 MAPKKK mediates TGF-β signaling by targeting SnoN oncoprotein for degradation. J. Biol. Chem. 282(13): 9475-81. Medline abstract: 17276978
Kobayashi, N., et al. (2007). c-Ski activates MyoD in the nucleus of myoblastic cells through suppression of histone deacetylases. Genes Cells. 12(3): 375-85. Medline abstract: 17352741
Krakowski, A. R., et al. (2005). Cytoplasmic SnoN in normal tissues and nonmalignant cells antagonizes TGF-β signaling by sequestration of the Smad proteins. Proc. Natl. Acad. Sci. 102(35): 12437-42. Medline abstract: 16109768
Leong, G. M., et al. (2001). Ski-interacting protein interacts with Smad proteins to augment transforming growth factor-β-dependent transcription. J. Biol. Chem. 276(21): 18243-8. Medline abstract: 11278756
Luo, K., et al. (1999). Ski interacts with the Smad proteins to repress TGFβ signaling. Genes Dev. 13: 2196-2206. Medline abstract: 10485843
Mizuhara, E., et al. (2005). Corl1: a novel neuronal lineage-specific transcriptional corepressor for the homeodomain transcription factor Lbx1. J. Biol. Chem. 280: 3645-3655. Medline abstract: 15528197
Nagata, M., et al. (2006). Nuclear and cytoplasmic c-Ski differently modulate cellular functions. Genes Cells. 11(11): 1267-80. Medline abstract: 17054724
Newfeld, S. and Wisotzkey, R. (2006). Molecular evolution of Smad proteins, pp 15-35 in Smad Signal Transduction: Smads in Proliferation, Differentiation and Disease, edited by C.-H. Heldin and P. Ten Dijke. Springer-Verlag, Dordrecht, The Netherlands.
Nomura, N., et al. (1989). Isolation of human cDNA clones of Ski and Sno. Nucleic Acids Res. 17: 5489-5500. Medline abstract: 2762147
Nomura, T., et al. (1999) Ski is a component of the histone deacetylase complex required for transcriptional repression by Mad and thyroid hormone receptor. Genes Dev. 13: 412-423. Medline abstract: 10049357
Nomura, T., et al. (2004). Oncogenic activation of c-Myb correlates with a loss of negative regulation by TIF1β and Ski. J. Biol. Chem. 279(16): 16715-26. Medline abstract: 14761981
Pearson-White, S., and Crittenden, R. (1997). Proto-oncogene Sno expression, alternative isoforms and immediate early serum response. Nucleic Acids Res. 25: 2930-2937. Medline abstract: 9207045
Pearson-White, S., and McDuffie, M. (2003). Defective T-cell activation is associated with augmented TGFβ sensitivity in mice with mutations in Sno. Mol. Cell. Biol. 23: 5446-5459. Medline abstract: 12861029
Pessah, M., et al. (2002). c-Jun associates with the oncoprotein Ski and suppresses Smad2 transcriptional activity. J. Biol. Chem. 277(32): 29094-29100. Medline abstract: 12034730
Ramel, M. C., et al. (2006). Drosophila SnoN modulates growth and patterning by antagonizing TGF-β signalling. Mech. Dev. 124(4): 304-17. Medline abstract: 17289352
Ramel, M. C., et al. (2007). Drosophila SnoN modulates growth and patterning by antagonizing TGF-beta signalling. Mech. Dev. 124(4): 304-17. PubMed citation
Sarker, K. P., Wilson, S. M. and Bonni, S. (2005). SnoN is a cell type-specific mediator of transforming growth factor-β responses. J. Biol. Chem. 280(13): 13037-46. Medline abstract: 15677458
Shinagawa, T., et al. (2000). The Sno gene, which encodes a component of histone deacetylase complex, acts as a tumor suppressor in mice. EMBO J. 19: 2280-2291. Medline abstract: 10811619
Shravage, B. V., Altmann, G., Technau, M. and Roth, S. (2007). The role of Dpp and its inhibitors during eggshell patterning in Drosophila. Development 134(12): 2261-71. Medline abstract: 17507396
Stegmuller, J., et al. (2006). Cell-intrinsic regulation of axonal morphogenesis by the Cdh1-APC target SnoN. Neuron 50(3): 389-400. Medline abstract: 16675394
Stroschein, S., Wang, W. Zhou, S., Zhou, Q. and Luo, K. (1999). Negative feedback regulation of TGFβ signaling by the SnoN oncoprotein. Science 286: 771-774. Medline abstract: 10531062
Stroschein, S., Bonni, S., Wrana, J. and Luo, K. (2001). Smad3 recruits the anaphase-promoting complex for ubiquitination and degradation of SnoN. Genes Dev. 15: 2822-2836. Medline abstract: 11691834
Suzuki, H., et al. (2004). c-Ski inhibits the TGF-β signaling pathway through stabilization of inactive Smad complexes on Smad-binding elements. Oncogene 23(29): 5068-76. Medline abstract: 15107821
Takaesu, N., et al. (2005). DNA-binding domain mutations in Smad genes yield dominant negative proteins or a neomorphic protein that can activate Wg target genes in Drosophila. Development 132: 4883-4894. Medline abstract: 16192307
Takaesu, N. T., et al. (2006). dSno facilitates baboon signaling in the Drosophila brain by switching the affinity of Medea away from Mad and toward dSmad2. Genetics 174(3): 1299-313. Medline abstract: 16951053
Takeda, M., et al. (2004). Interaction with Smad4 is indispensable for suppression of BMP signaling by c-Ski. Mol. Biol. Cell 15(3): 963-72. Medline abstract: 14699069
Ueki, N., Zhang, L. and Hayman, M. J. (2004). Ski negatively regulates erythroid differentiation through its interaction with GATA1. Mol. Cell. Biol. 24(23): 10118-25. Medline abstract: 15542823
Wrighton, K. H., et al. (2007). Transforming growth factor-β-independent regulation of myogenesis by SnoN sumoylation. J. Biol. Chem. 282(9): 6517-24. Medline abstract: 17202138
Wu, J., et al. (2002). Structural mechanism of Smad4 recognition by the nuclear oncoprotein Ski: insights on Ski-mediated repression of TGFβ signaling. Cell 111: 357-367. Medline abstract: 12419246
Wu, K.. et al. (2003). DACH1 inhibits TGF-β signaling through binding Smad4. J. Biol. Chem. 278: 51673-51684. 14525983
Yoshida, S., et al. (2005). Dpp signaling controls development of the lamina glia for retinal axon targeting in the visual system. Development 132: 4587-4598. Medline abstract: 16176948
Zhu, Q., Pearson-White, S. and Luo, K. (2005). Requirement for the SnoN oncoprotein in transforming growth factor β-induced oncogenic transformation of fibroblast cells. Mol. Cell. Biol. 25(24): 10731-44. Medline abstract: 16314499
Zhu, Q., et al. (2007). Dual role of SnoN in mammalian tumorigenesis. Mol Cell Biol. 27(1): 324-39. Medline abstract: 17074815
date revised: 15 March 2007
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