dSno overexpression antagonizes BMP signaling:

Studies in mammalian cells showed that, when overexpressed, Sno is an antagonist of TGFß/Activin signaling (e.g., Luo, 1999). Overexpression of dSno with A9.Gal4 (throughout the presumptive wing blade) resulted in small wings with multiple vein truncations at 100% penetrance. A9.Gal4:UAS.dSno pupal wing discs were examined for Drosophila serum response factor (dSRF) expression, an intervein marker repressed by Dpp signaling. In A9.Gal4:UAS.dSno pupal discs, dSRF expression is highly irregular with no obviously downregulated regions corresponding to vein primordia. These wing and disc phenotypes are strongly reminiscent of those expressing the dominant-negative allele Mad1 (DNA binding defective but competent to bind Medea) with a variety of drivers, including A9.Gal4 (100% penetrant) and 69B.Gal4. Mad1 dominant-negative effects are due to the titration of Medea into nonfunctional complexes (Takaesu, 2005). The similarity of dSno and Mad1 phenotypes suggests that overexpression of dSno antagonizes BMP signaling (Takaesu, 2006).

This was further tested this by coexpressing dSno with Medea or Mad or dSmad2. Coexpression of dSno with Medea or Mad rescues the dSno phenotype to nearly wild type in size and vein pattern. In dSno and Medea coexpressed wings, reduced size was completely eliminated and multiple vein defects were reduced to 28% penetrance. In dSno and Mad coexpressed wings, reduced size was completely eliminated and multiple vein defects were reduced to 19% penetrance. Alternatively, coexpression of dSno with dSmad2 has little effect on the dSno phenotype. In dSno and dSmad2 coexpressed wings, reduced size and multiple vein defects remained 100% penetrant. Coexpression of Mad1 and dSno significantly enhanced the dSno phenotype. One hundred percent of Mad1 and dSno coexpressing the wings are more abnormal than those expressing either dSno or Mad1 (Takaesu, 2005). The coexpressing wing is very small and veinless and resembles wings expressing UAS.Dad (Dpp antagonist) or dpp class II disc mutants (e.g., dppd5). The enhanced phenotype suggests that dSno and Mad1 antagonize BMP signaling in distinct ways that have additive effects (Takaesu, 2006).

Experiments with a constitutively activated form of the Dpp type I receptor Thickveins (CA-Tkv) are also consistent with this hypothesis. One hundred percent of A9.Gal4:UAS.CA-Tkv wings are overgrown and have numerous ectopic veins as well as vein truncations. This phenotype is suppressed in 98% of the individuals when UAS.dSno is coexpressed with UAS.CA-Tkv. In fact, the coexpression phenotype is not much different from A9.Gal4:UAS.dSno alone, indicating that dSno antagonism of Dpp signaling is fully epistatic to activated Tkv. Finally, ubiquitous overexpression of dSno in the embryonic ectoderm with 32B.Gal4 resulted in discless larvae—a phenotype seen in Mad and Medea null genotypes and in dpp class V disc mutants ( e.g., dppd12. It is concluded that overexpression of dSno antagonizes BMP signaling (Takaesu, 2006).

dSno promotes the interaction of Medea with dSmad2

To resolve the apparent contradiction between the effect on signaling of dSno overexpression (antagonism) and the role identified in dSno mutants (mediation), dSno was examined biochemically. Expression constructs encoding Flag or T7 epitope-tagged Medea, Mad, and dSmad2 were used and various combinations were co-expressed in COS1 cells. It was possible to clearly detect interaction of Medea with both dSmad2 and Mad by co-immunoprecipitation. A T7-tagged dSno expression construct was generated and coexpressed with Flag-dSmad2, Mad, or Medea or with a control vector. Complexes were isolated on Flag agarose and analyzed for the presence of coprecipitating dSno by T7 Western blot. T7-dSno was readily detectable in complexes isolated from cells expressing Medea, but not Mad or dSmad2 (Takaesu, 2006).

Since Medea is a shared partner for both Mad and dSmad2, whether co-complexes containing Medea and dSno together with either Mad or dSmad2 was tested. COS1 cells were transfected with T7-dSno and Flag-Mad or dSmad2 with or without T7-Medea. T7-dSno was present in a complex with Flag-dSmad2 only when T7-Medea was also present. Interestingly, approximately equal amounts of Medea and dSno appeared to coprecipitate with dSmad2, suggesting that much of the Medea that interacts with dSmad2 in this assay is also bound to dSno. In contrast, dSno was not detected in complex with Mad, even when Medea was present, even though Medea clearly interacted with Mad in this assay. These results suggest that dSno interacts specifically with Medea and that the dSno-Medea complex can interact with dSmad2 but not with Mad (Takaesu, 2006).

To test whether incorporation of dSno affected formation of the Medea-dSmad2 complex, Flag-Medea and T7-dSmad2 were coexpressed with or without dSno. The amount of dSmad2 that coprecipitated with Flag-Medea was clearly increased in the presence of dSno. In the reverse of this experiment, it was also observed that an increase in T7-tagged Medea present in Flag-dSmad2 precipitates when dSno was coexpressed. These results suggest that dSno may promote the formation of Medea-dSmad2 complexes (Takaesu, 2006).

To test whether dSno has any effect on the formation of Mad-Medea complexes, similar experiments were performed in which Flag-Medea and Western blotted was isolated for coprecipitating T7-Mad or dSmad2 in the presence or absence of coexpressed dSno. The interaction of Medea with Mad was more readily detectable than with dSmad2. However, inclusion of dSno again increased the interaction between Medea and dSmad2. In contrast, no increase was seen in the Medea–Mad interaction when dSno was coexpressed and it appeared that increasing dSno expression decreased the amount of Mad that coprecipitated with Flag-Medea. Thus it appears that dSno not only may promote interaction of Medea with dSmad2, but also may compete with Mad for Medea interaction, suggesting that dSno may play a role in determining the pathway specificity of Medea (Takaesu, 2006).

snoN: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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