Supplemental Figure 1
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Fig.S1. Dl and Ser
expression in third instar wing disks. Disks carrying C253-Gal4; UAS-srcEGFP (green), which marks the proneural
clusters with a membrane-targeted GFP, were stained with anti-Delta (red) and
anti-Serrate (blue). All disks are oriented with dorsal/proximal upwards and
anterior towards the left. Scale bars: 40 mm. A¢-D¢ are the Dl channels; A¢¢-D¢¢ are the
Serrate channels of A-D, respectively. (A) Mid-third instar disk, where Ser
expression is mostly dorsal (A¢¢) and many proneural clusters have not yet formed. (B-D) Late third
instar disks (with a leg disk at the left in B). (C) Detail of a late wing
pouch region; (D) detail of a notum. Some proneural clusters are marked with
their names: L3, DC, SA, PA/tr1, NP. In the wing, Ser is exclusively dorsal
early on (A¢¢) and
maintains a dorsal bias throughout larval life (B¢¢,C¢¢). By late third instar, both DSL proteins are expressed in two stripes
flanking the wing margin and along the longitudinal veins L3, 4 and 5; dorsal
L3 overlaps with a proneural cluster (B,C). In the notum, Dl and Ser are each
expressed in a wide stripe along the proximodistal axis, which resolves into a
U-shaped pattern (D). The Ser expression domain is slightly displaced to the
posterior of the Dl domain. Three proneural clusters, dorsocentral (DC),
notopleural (NP) and supra-alar (SA), are located in the region of overlap
between the Dl and Ser U-shaped swaths. Proneural clusters are not always found
in regions of high Dl/Ser accumulation, e.g. the PA/tr1 proneural cluster is
just posterior to the U-shaped swaths, in a region of background Dl/Ser
expression. It is possible that such background levels represents real
expression across the wing disk, because it is diminished further in the
respective mutant mosaic backgrounds (Dl or Ser; see Fig. S3A,C). Arrowheads indicate SOPs,
where Dl and Ser accumulation is downregulated (better examples can be seen in
Fig. 3B¢,B¢¢,D¢,D¢¢ colored arrowheads).
Supplemental Figure 2
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Fig.S2. Nota mosaic for
Dl Ser with
simultaneous uniform expression of UAS-Dl (A) or UAS-Ser (B). Mutant cells express GFP. (A) Dl staining
(red, shown separately in A¢) shows the level of Dl overexpression – regions of overexpression
(green arrow) were compared by densitometry with regions of high endogenous
expression (red arrow). (B) Ser staining (red, shown separately in B¢) shows the level of Ser
overexpression; arrows as in A¢.
Supplemental Figure 3
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Fig.
S3. Dl and Ser are
internalized by Neur independently of each other. All wing disks are oriented
dorsal upwards and anterior towards the left. In all panels, Ser is red (shown
separately in A¢-D¢) and Dl is blue (shown separately
in A¢¢-D¢¢). (A) Ser mutant cells are marked by absence of cytoplasmic
GFP (green). Even the low levels of Ser ventrally are further diminished in
mutant tissue (A¢, asterisk). By
contrast, Dl expression within the clone is not affected (other than a
reduction at the wing margin, where N signaling is known to positively regulate
DSL gene expression and the large Ser clone has compromised N signaling); arrows in
A¢¢ indicate Dl expression within the Ser clone. (B) Ser mutant cells simultaneously express
UAS-neur and UAS-GFP; therefore the presence of GFP
marks mutant territories. As Dl and Ser are membrane proteins and GFP is
nuclear, the clone boundaries are not perfectly matched and are better
visualized in the red (Serrate) channel. Unlike A, mutant territory in this
disk shows strong downregulation of Dl (B¢¢, arrows), which is due to the expression of
Neur. (C) Clones mutant for Dl (marked by the absence of GFP, green) lack Dl staining (C¢¢) but do not affect Ser expression
(C¢, arrows). (D) Clones mutant for Dl and simultaneously expressing UAS-neur and UAS-GFP (clones are positively marked with
GFP, green). Unlike C, areas that lack Dl, also downregulate Ser (D¢, arrows), owing to the expression
of Neur. From A and C, we conclude that removal of either DSL protein does not
affect the expression of the other. From B and D we conclude that Neur can
downregulate the levels of both Dl and Ser in the absence of the other DSL
protein. Colored arrowheads in B¢, B¢¢, D¢ and D¢¢ are examples of SOPs in wild-type territory,
which exhibit downregulation of both Dl (blue arrowheads) and Ser (red
arrowheads). This is probably owing to endogenous neur expression.
