Document S1. Four Figures and Supplemental Experimental

Current Biology, Volume 20
Supplemental Information
A Role of Receptor Notch
in Ligand cis-Inhibition in Drosophila
Isabelle Becam, Ulla-Maj Fiuza, Alfonso Martínez Arias, and Marco Milán
Figure S1. Notch Protein Levels in Different Mutant Conditions
(A) Wing discs with clones of cells expressing GFP (green) and NdsRNA and labelled to visualize Dl protein
in purple or white.
(B and C) Wing discs with clones of cells marked by the presence (B) or absence (C) of GFP (green) and
labeled to visualize in red or white the extracellular or intracellular domains of Notch. Scale bars
represent 10 μm.
Figure S2. Consequences of Expressing NdsRNA in Clones of Cells
(A-D) Wing (A-C) and leg (D) imaginal discs with clones of cells expressing GFP (green) and NdsRNA and
labelled to visualize msh-lacZ (antibody to β-gal, red or white, A), Cut (red or white, B), wg-lacZ (antibody
to β-gal, red or white, C) or Ser (purple of white) expression. Note cell autonomous loss of Cut or wg-lacZ
expression (green arrowheads), non-autonomous induction of Cut or wg-lacZ expression (red arrows) and
increase in Ser protein levels (pink arrows) in these clones. d, dorsal, and v, ventral compartments.
Higher magnifications of boxed fields are shown in B, C. Scale bars represent 10 μm.
(E) Notch effects on ligand signaling activity. S2 Mt-N cells were co-transfected with Fringe (Fng), a
luciferase Notch reporter and a control pol III-Renilla luciferase to assess Notch signaling activity. These
cells were co-cultured with S2 cells expressing either Ser, or Ser and Notch, or an empty vector as a
control. The data presented are the average of 3 replicates (±SD) and are from one experiment
representative of 3 independent experiments.
Figure S3. Localization of Ser in Endocytic Vesicles and Analysis of the Notch Extracellular
Domain in Ser cis-Inactivation
(A and C-E) Sub-cellular localization of Ser (red) in the endocytic compartment of wing cells abutting the
DV boundary. White arrowheads in A, C, D or red circles in E point to Ser containing vesicles labelled by
Rab5-GFP (A, green), Rab7-GFP (D, green) or Rab11-GFP (E, green) containing vesicles or vesicles
labelled with Rab5 antibodies (C, green).
(B) Rab5-GFP (red) containing vesicles were labelled with rabbit anti-Rab5 antibodies (green).
(F-J) Wing discs with clones expressing GFP, NdsRNA and Necn (F, G) or mutant for NAxM1 and marked by
the absence of GFP (H-J). Wing discs were labeled to visualize the extracellular domain of Notch (F, in
blue and H, in purple), Ser protein levels (F, I, in red or white) and Wg (G, red or white) or Cut (J, in blue
or white) protein expression.
(K) Histogram plotting the number of Ser containing vesicles observed in 8 NCo mutant clones (red bars)
when compared to wild-type abutting cells (blue bars). Scale bars represent10 μm.
Figure S4. Model depicting trans and cis Interactions between Notch and Its Ligands during DV
Boundary Formation in the Drosophila Wing
(A) Notch-ligand interactions in trans between signal-sending (red) and signal-receiving (green) cells
induce Notch activation (grey arrows) along the DV boundary. Interactions in cis between Notch and its
ligands inhibit Notch in a cell-autonomous manner and limit the levels of Ser ligand at the cell surface,
thus preventing Notch activation in the signal-sending cell population.
(B) In the absence of Notch (grey cell), cell surface levels of Serrate (Ser, depicted in blue), but not Delta
(Dl, depicted in red), increase and induce Notch activation (grey arrows) in the signal-sending population.
(C) In the absence of ligand expression (grey cell), Notch is activated (grey arrows) in the signal sending
population in a cell-autonomous manner by the activity of the ligands expressed in neighboring cells.
Supplemental Experimental Procedures
Fly Strains
Ubi-Rab5-YFP [1]; Tub-Rab7-GFP[2]; Tub-Rab11-YFP [3]; Ser-lacZ [4]. UAS-NdsRNA, an inducible
Notch hairpin construct for RNAi, using the Notch coding sequence contained in exon 6 [5]; kuz1405 [6];
D-mib1and D-mib2 [7], actin>CD2>GAL4 (AFG in the text, [8]); UAS- NINTRAand UAS- NNEXT [9]; AxM1
[10], N55e11[11], NCo [12], NM1 [13] are described in Flybase. The Drosophila genotypes used to
generate genetic mosaics are described in Supplemental Data.
Antibodies
Antibodies against the following proteins are described in the Developmental Studies Hybridoma Bank:
Notch extracellular domain (C458.2H, [14]), Notch intracellular domain (C17.9C6, [15]), Cut (2B10, [16]),
Delta (C594.9B, [17]) and Wg (4D4, [18]). β-Galactosidase (Cappel). Rat anti-Serrate [19]. Rabbit antiRab5 (Abcam, ab31261) was used at 1:250 dilution. Endocytic vesicles expressing Rab5-GFP [1] were
labelled with rabbit anti-Rab5 (Figure S3C)
Genetic Mosaics
The following Drosophila genotypes were used to generate:
(a) loss-of-function clones by the FLP/FRT system [20]:
NCo FRT18A/arm-lacZ FRT18A; hs-FLP (II)
NM1 FRT101/UbiGFP FRT101; ptc-Gal4 UAS-FLP
N55e11 FRT19A/ Gal80 FRT19A; hs-FLP tub-Gal4 UAS-GFP
N55e11 FRT19A/ Ubi-GFP FRT19A; Ubx-FLP
AxM1 FRT101/UbiGFP FRT101; ptc-Gal4 UAS-FLP
hs-FLP; kuz1405 FRT40A/ UbiGFP FRT40A
(b) loss-of-function clones by the MARCM (mosaic analysis with a repressible cell marker) technique to
simultaneously express diverse transgenes in the clones [21]:
hs-FLP tub-Gal4 UAS-GFP/UAS NdsRNA; FRT82B / FRT82B Gal80
hs-FLP tub-Gal4 UAS-GFP/UAS NdsRNA; FRT82B DlRvF10 SerRX82/ FRT82B Gal80
hs-FLP tub-Gal4 UAS-GFP/UAS-NdsRNA; FRT82B DlRvF10 / FRT82B Gal80
hs-FLP tub-Gal4 UAS-GFP/UAS-NdsRNA; FRT82B SerRX82/ FRT82B Gal80
hs-FLP tub-Gal4 UAS-GFP; D-mib11 FRT2A / Gal80 FRT2A
hs-FLP tub-Gal4 UAS-GFP; D-mib12 FRT2A / Gal80 FRT2A
hs-FLP tub-Gal4 UAS-GFP/UAS-NdsRNA; D-mib11 FRT2A / Gal80 FRT2A
(c) clones of cells expressing different transgenes:
hs-FLP/UAS-NdsRNA; Actin>CD2>Gal4 UAS-GFP
hs-FLP/UAS-NdsRNA; Actin>CD2>Gal4 UAS-GFP/ UAS-NINTRA
hs-FLP; Actin>CD2>Gal4 UAS-GFP/ UAS-NINTRA
hs-FLP/UAS-NdsRNA; Actin>CD2>Gal4 UAS-GFP/ UAS-NNEXT
hs-FLP/UAS-NdsRNA; Actin>CD2>Gal4 UAS-GFP/ UAS-NECN
hs-FLP; Actin>CD2>Gal4 UAS-GFP/ UAS-NNEXT
Clone induction by heat-shock was carried out 2-3 days before dissection of larval discs. Larvae were
grown at 25ºC. Under these circumstances, clones did not violate the DV boundary (Figure S2) and the
DV topological location of the clones could be easily determined. Notch protein levels were monitored in
all these clones by using two distinct antibodies against the intracellular and extracellular domains of
Notch (Fig. S1B).
Quantification of Endocytic Vesicles in Fixed Tissue
The number of Ser containing vesicles was quantified in 16 N55e11, 7 D-mib12 and 8 Nco mutant clones
and in nearby wild-type regions of the same size. Scored clones were located close to the DV boundary
and the nearby wilt type regions were located at the same distance to the DV boundary as the clones.
Histograms were plotted with Microsoft Excel, where the numbers of Ser containing vesicles in mutant
and wild-type territories were presented as pairs. Using Microsoft-Excel Software, t-test analysis was
carried out.
Endocytosis Assay
As previously described [7], third instar larvae wing discs were dissected in Schneider's Drosophila
medium (Gibco BRL, San Diego, California, United States) containing 10% fetal calf serum (Gibco BRL).
Wing discs were cut between the wing pouch and the thorax to facilitate antibody diffusion. Wing discs
were cultured for 15 min with rat anti-Ser antibody 1/50 [19]. Following three medium changes and a 20min chase period, wing discs were fixed and incubated with secondary antibodies.
Plasmids and Constructs
pMT-Dl was from the Drosophila Genomics Resources Centre (DGRC). pMT-Fng and pMT-Notch-HA
[22]. pMT-Ser was provided by François Schweisguth. The Notch reporter construct is described in [22].
pol III Renilla luciferase [23].
Luciferase Assays
Signaling in S2 Mt-N cells (Notch stable cell line from the DGRC) was induced by co-culture with an equal
amount of ligand-expressing cells (S2 Mt-Dl cells from the DGRC or S2 cells transiently transfected with
0.1 μg of pMT-Dl, with 0.1 μg of pMT-Ser or/and with 0.1 μg of 0.1 pMT-Notch-HA). Reporter cells were
transfected with 0.025 μg of the reporter plasmid (NRE), 0.025 μg of polII-Renilla Luciferase and the
amount of pMT-Fng used in transfections was of 0.1 μg. Equal amounts of DNA were used in the
transient transfections by adjusting with an empty vector. Cells were transiently transfected using the
Qiagen Effectene kit, washed twice and then co-cultured for 48 h with 0.7 mM CuSO4 induction. The cell
assays were made in triplicate wells for each condition and three independent experiments were done for
each assay. The data presented are from one experiment representative of three independent
experiments. The values presented are averages of the three replicates normalized to Renilla luciferase
to correct for transfection efficiency and presented as the fold change in activity to ligand-induced Notch
signaling activity. Luciferase activity was measured using the Dual-Glo system from Promega. p values
were calculated with the Microsoft Excel Function of two-sample t-test assuming unequal variances.
Plasmids and constructs are described as Supplemental Data.
Supplemental References
1.
Zhang, J., Schulze, K.L., Hiesinger, P.R., Suyama, K., Wang, S., Fish, M., Acar, M., Hoskins,
R.A., Bellen, H.J., and Scott, M.P. (2007). Thirty-one flavors of Drosophila rab proteins. Genetics
176, 1307-1322.
2.
Entchev, E.V., Schwabedissen, A., and Gonzalez-Gaitan, M. (2000). Gradient formation of the
TGF-beta homolog Dpp. Cell 103, 981-991.
3.
Classen, A.K., Anderson, K.I., Marois, E., and Eaton, S. (2005). Hexagonal packing of Drosophila
wing epithelial cells by the planar cell polarity pathway. Dev Cell 9, 805-817.
4.
Yan, S.J., Gu, Y., Li, W.X., and Fleming, R.J. (2004). Multiple signaling pathways and a selector
protein sequentially regulate Drosophila wing development. Development 131, 285-298.
5.
Presente, A., Shaw, S., Nye, J.S., and Andres, A.J. (2002). Transgene-mediated RNA
interference defines a novel role for notch in chemosensory startle behavior. Genesis 34, 165169.
6.
Sotillos, S., Roch, F., and Campuzano, S. (1997). The metalloprotease-disintegrin Kuzbanian
participates in Notch activation during growth and patterning of Drosophila imaginal discs.
Development 124, 4769-4779.
7.
Le Borgne, R., Remaud, S., Hamel, S., and Schweisguth, F. (2005). Two distinct E3 ubiquitin
ligases have complementary functions in the regulation of delta and serrate signaling in
Drosophila. PLoS Biol 3, e96.
8.
Pignoni, F., and Zipursky, S.L. (1997). Induction of Drosophila eye development by
decapentaplegic. Development 124, 271-278.
9.
Struhl, G., and Adachi, A. (1998). Nuclear access and action of Notch in vivo. Cell 93, 649-660.
10.
de Celis, J.F., and Garcia-Bellido, A. (1994). Modifications of Notch function by Abruptex
mutations in Drosophila melanogaster. Genetics 136, 183-194.
11.
de Celis, J.F., and Garcia Bellido, A. (1994). Roles of the Notch gene in Drosophila wing
morphogenesis. Mech. Devel. 46, 109-122.
12.
Lyman, D., and Young, M.W. (1993). Further evidence for function of the Drosophila Notch
protein as a transmembrane receptor. Proc Natl Acad Sci U S A 90, 10395-10399.
13.
de Celis, J.F., Barrio, R., del Arco, A., and Garcia-Bellido, A. (1993). Genetic and molecular
characterization of a Notch mutation in its Delta and Serrate binding domain in Drosophila. Proc
Natl Acad Sci 90, 4037-4041.
14.
Diederich, R.J., Matsuno, K., Hing, H., and Artavanis-Tsakonas, S. (1994). Cytosolic interaction
between deltex and Notch ankyrin repeats implicates deltex in the Notch signalling pathway.
Development 120, 473-481.
15.
Fehon, R.G., Kooh, P.J., Rebay, I., Regan, C.L., Xu, T., Muskavitch, M.A.T., and ArtavanisTsakonas, S. (1990). Molecular interactions between the protein products of the neurogenic loci
Notch and Delta, two EGF-homolgous genes in Drosophila. Cell 61, 523-534.
16.
Blochlinger, K., Bodmer, R., Jan, L.Y., and Jan, Y.N. (1990). Patterns of expression of cut, a
protein required for external sensory organ development in wild type and cut mutant Drosophila
embryos. Genes Dev 4, 1322-1331.
17.
Qi, H., Rand, M.D., Wu, X., Sestan, N., Wang, W., Rakic, P., Xu, T., and Artavanis-Tsakonas, S.
(1999). Processing of the notch ligand Delta by the metalloprotease Kuzbanian. Science 283, 9194.
18.
Brook, W.J., and Cohen, S.M. (1996). Antagonistic Interactions Between Wingless and
Decapentaplegic Responsible for Dorsal-Ventral Pattern in the Drosophila Leg. Science 273,
1373-1377.
19.
Weihe, U., Milan, M., and Cohen, S.M. (2001). Regulation of Apterous activity in Drosophila wing
development. Development 128, 4615-4622.
20.
Xu, T., and Rubin, G.M. (1993). Analysis of genetic mosaics in developing and adult Drosophila
tissues. Development 117, 1223-1237.
21.
Lee, T., and Luo, L. (1999). Mosaic analysis with a repressible cell marker for studies of gene
function in neuronal morphogenesis. Neuron 22, 451-461.
22.
Krejci, A., and Bray, S. (2007). Notch activation stimulates transient and selective binding of
Su(H)/CSL to target enhancers. Genes Dev 21, 1322-1327.
23.
Hayward, P., Brennan, K., Sanders, P., Balayo, T., DasGupta, R., Perrimon, N., and Martinez
Arias, A. (2005). Notch modulates Wnt signalling by associating with Armadillo/beta-catenin and
regulating its transcriptional activity. Development 132, 1819-1830.

Download Report