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First published online 16 March 2005
doi: 10.1242/dev.01724

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Notch modulates Wnt signalling by associating with Armadillo/ß-catenin and regulating its transcriptional activity

¹ Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK
² School of Biological Sciences, University of Manchester, 3.239 Stopford Building, Oxford Road, Manchester M13 9PT, UK
³ Harvard Medical School/HHMI, Department of Genetics, 77 Avenue Louis Pasteur, NRB#339, Boston, MA 02115, USA

View larger version (104K): [in a new window]   Fig. 1. Notch modulates the activity of an activated form of Armadillo. (A-D) Apical sections through third larval instar wing discs stained for endogenous Armadillo (N27A1) and expressing different signalling molecules under the control of dpp GAL4. (A) Wild-type wing disc. Notice elevated levels of Armadillo around the dorsal-ventral boundary (arrow) which coincide with high levels of Wingless signalling. The white line indicates the region of ectopic expression in experimental situations. (B) Disc expressing TNotch. Slight differences in the pattern of endogenous Armadillo, particularly at the DV boundary are observed. (C) Wing disc expressing Armadillos10 (Pai et al., 1997). This molecule lacks the epitope recognised by the monoclonal antibody N27A1. The domain of expression of ArmadilloS10 expression is demarcated by changes in the concentration and subcellular location of the endogenous Armadillo. Also note the alterations of growth in the notum (arrowhead) (see Pai et al., 1997). (D) Co-expression of TNotch with ArmadilloS10 suppresses significantly the effects of ArmadilloS10 both on the shape of the disc and on the altered distribution of endogenous Armadillo (compare to C, similar focal plane, also see Fig. S1 in supplementary material). (E-G) Wing disc expressing ArmadilloS10, apical section at the level of the adherens junctions; posterior to right and anterior to left. (E) ArmadilloS10 detected with anti-myc antibody. Notice its association with the adherens junctions. (F) Endogenous Armadillo (N27A1 antibody) is excluded from the adherens junctions over the domain of ArmadilloS10 expression. The shadows correspond to the cells from the peripodial membrane. (G) Merged image of E and F.

View larger version (169K): [in a new window]   Fig. 2. Notch suppresses the activity of ArmadilloS10. Wing pouch region of third larval instar wing discs showing the response of high (Senseless, Sens) and low (Distalless, Dll) threshold targets of Wingless signalling to normal or ectopic activity of Armadillo. (A-C) Wild type. (A) Distalless expression; notice a slight elevation of the expression at the DV boundary. The white line indicates the approximate domain of dpp expression. (B) Senseless expression highlighting neural precursors that develop in response to high levels of Wingless signalling (Couso et al., 1993). (C) Merged image of A and B. (D-F) Wing pouch of a disc expressing ArmadilloS10 under the control of dppGAL4. (D) Distalless expression is now elevated and expanded over the AP boundary. (E) Senseless can be detected over a new domain along the AP boundary. (F) Merged image of D, E. (G-I) Wing pouch of a disc expressing ArmadilloS10 and TNotch under the control of dppGAL4. (G) The effects of ArmadilloS10 on Distalless are suppressed by TNotch, a reduction of wild-type levels is also observed. (H) The ectopic expression of Senseless induced by ArmadilloS10 is suppressed by TNotch. Notice also the reduction in the endogenous expression over the domain of TNotch expression. (I) Merged image of G and H.

View larger version (70K): [in a new window]   Fig. 3. Notch affects the levels of Armadillo and of ArmadilloS10. (A-D) Effects of TNotch on the stability of ArmadilloS10 and wild-type Armadillo in third instar wing discs (anterior down and posterior up); expression is under the control of dppGAL4 at 22°C. (A) Expression of ArmadilloS10. (B) Expression of TNotch with ArmadilloS10; notice the reduction in the overall amount of ArmadilloS10. (C) Expression of wild-type Armadillo. Armadillo (red) is very unstable and is only stabilised in the presence of Wingless (green; see also Fig. S3 in supplementary material). (D) Expression of wild-type Armadillo with TNotch. The ectopic Armadillo has been eliminated except for a small amount in the neighbourhood of the Wingless-expressing cells (see inset) and this is not associated with a loss of Wingless expression. (E,F) Western blots showing the concentration of endogenous Armadillo and ArmadilloS10 in the presence or absence of various forms of Notch, expression is under the control of dppGal4 (F) or C5Gal4 (E). In the presence of Armadillos10 an elevation of endogenous Armadillo levels (E) or an increase in the hypophosphorylated form (lower band of doublet, F) is observed compared to wild type. Expression of TNotch under the control of dppGal4 results in a marked decrease of both Armadillo and Armadillos10 (due to insufficient separation Armadillo doublet is visualised as one band in E), whereas expression of TNotch under the control of C5Gal4 results in a reduction of hypophosphorylated form of endogenous Armadillo and of Armadillos10 (F). The effects of full length Notch (Notch) are less marked, under the control of dppGal4 Notch expression results in a decrease of endogenous Armadillo (E); and with C5Gal4 a reduction in Armadillos10 is apparent. (F). Expression of NICD results in small increases in the amounts of Armadillos10 (E,F).

View larger version (33K): [in a new window]   Fig. 4. Notch modulates Wnt pathway transcriptional activity in both Drosophila and vertebrate cells. (A) Diagram of the Notch molecules used. (B) Ectopic activation of the Wnt signalling pathway was observed in Drosophila clone8 (cl8) cells in the presence of Notch dsRNA (104-fold activation compared to no dsRNA), but not GFP dsRNA (1.1-fold activation). (C,D) In Drosophila SL2 (C), or S2R+ cells (D) Wnt signalling was induced with an oncogenic form of ß-catenin, S37A ß-catenin (Schweizer and Varmus, 2003), the presence of a membrane tethered form of Notch (TNotch) significantly reduced the level of ectopic Wnt signalling (C,D). (E-H) N-N1 (delN-N1) cleaves spontaneously to release the NICD domain of Notch1 as shown by the strong activation of the CBF1 reporter (H), whereas LNR-N1 rarely cleaves as shown by the weak activation of the CBF1 reporter (H) (Mumm et al., 2000). A further inhibitor of Wnt signalling ExFz8 acts by titrating Wnt (Brennan et al., 2004). Ectopic Wnt signalling was induced with Wnt1, delN-LRP6, Dishevelled, activated ß-catenin or LEF1-VP16 in HEK-293T cells. Both forms of Notch are capable of significantly repressing ectopic Wnt signalling induced by Wnt1, Dsh, and activated ß-catenin (E,F), LNR-N1 effects extended to ectopic Wnt signalling induced by delN-LRP6 and LEF1-VP16. Whereas, ExFz8 repressed ectopic Wnt signalling induced by Wnt1, some small effects on the intracellular mediators of Wnt signalling were observed, such effects have previously been reported (Suzuki et al., 2004) (G).

View larger version (131K): [in a new window]   Fig. 5. Effects of loss of function of Notch and shaggy on the stability of Armadillo. (A,D,G,J) GFP, green; (B,E,H,K) endogenous Armadillo, red; (C,F,I,L) merge of GFP and Armadillo images. (A-C) Loss of function of shaggy (loss of GFP) results in a cell autonomous elevation of the levels of Armadillo, which remains largely associated with adherens junctions (inset C, apical is up). Notice that only clones of a certain size (> about five cells) show the elevated levels of Armadillo (arrowheads in A,B); this is probably due to the long perdurance of Shaggy. The epithelium looks very thick (compare to inset F) because the loss of shaggy affects the epithelial organisation of the cells (A.M.A., unpublished data). (D-F) Simultaneous loss of Notch and shaggy results in very elevated levels of Armadillo that appear delocalised within the cytoplasm (inset F). The clones are small. Loss of Notch function affects cell proliferation (de Celis and Garcia-Bellido, 1994). (G-I) Expression of wild-type Armadillo under the control of ptcGal4 results in a Wingless-dependent stabilisation of Armadillo (see also Fig. S3 and Fig. S4 in supplementary material) in a narrow band at the AP border and, in particular, at the intersection with the dorsoventral boundary (arrowhead in H) where levels of Wingless are highest. Clones of wild-type cells (loss of GFP) do not change the instability of the ectopic Armadillo (dots indicate regions in clones where Armadillo has not accumulated). (J-L) Wing disc heterozygous for Notch (Df(1)N81k/+). The ptcGal4-driven expression of Armadillo is broader and contains more cells maintaining higher levels of Armadillo than in wild type. Furthermore, within the Notch mutant clones an increased number of cells have high levels of Armadillo (compare with G-I). Notice some clones, that lie far from that AP boundary (arrowhead in J-L) maintain high levels of Armadillo. N.B. In general we do not observe changes in the levels of Armadillo as a result of loss of Notch function alone, but in some experiments we observe an elevation in the levels of Armadillo. This elevation is always observed in the neighbourhood of the DV boundary (A.M.A., unpublished data). Unfortunately this effect is not reproducible and therefore should remain anecdotal.

View larger version (106K): [in a new window]   Fig. 6. TNotch can suppress Wingless signalling induced by the loss of function of GSK3ß/Shaggy. (A-D) Wild-type wing disc harbouring clones of cells mutant for shaggy. (A,B) Loss of shaggy function (black in A,C) leads to ectopic expression of the high threshold target of Wingless signalling Senseless (B) and ectopic elevation of Armadillo (D). (E-H) Wing discs expressing TNotch under the control of dppGal4 and containing clones of cells lacking shaggy. TNotch reduces the ectopic expression of Senseless (F) and the elevation of Armadillo (G). H is a merged colour image of E-G. The effect on Senseless is fully penetrant but that on Armadillo can be variable (A.M.A., unpublished data). Anterior is to the left and posterior to the right.

View larger version (90K): [in a new window]   Fig. 7. Armadillo associates with Notch in Drosophila embryos. Notch protein was immunoprecipitated from wild-type embryos and the presence of associated proteins was assessed by western blot. The majority of Notch protein present in embryo lysate and immunoprecipitated was the full-length protein (>250 kDa) which, in Drosophila is the predominant form of Notch at the cell surface (Kidd and Lieber, 2002). Detected in association with immunoprecipitated Notch were both Armadillo (middle panel) and Dishevelled (upper panel) protein. The lane labelled `lysate' represents a fifth of the total protein added to the immunoprecipitation reaction. Immunoprecipitated Notch reflects about 5-10% of the total Notch and associated with this is 0.5-3% of the total Armadillo and less than 0.5% of the total Dishevelled. Given that the pool of Armadillo associated with Notch is not associated with E-cadherin it is not surprising that this Notch-associated fraction of Armadillo is only a small proportion of the total cellular Armadillo.

View larger version (20K): [in a new window]   Fig. 8. Modulation of Wnt signalling by Notch in Drosophila (see text for details of interactions). In the steady state, Armadillo exists in a number of molecularly distinct pools which appear to be in equilibrium. Armadillo associates readily with cadherin. Also it, associates with a complex, which includes Axin and APC, leading to its phosphorylation on the N terminus by GSK3ß (Shaggy) and the delivery of the phosphorylated form to the proteasome, where it is degraded. In addition, Axin can prevent the formation of its active complex with TCF in a GSK3ß-independent manner. Our results indicate that Notch modulates the activity and amounts of hypophosphorylated Armadillo either by targeting the GSK3ß-independent activity of Axin or via an independent mechanism. The net effect of the inactivation of Notch and the Axin-based complex results in an efficient accumulation of Armadillo in the nucleus and its interaction with TCF.