Uif, a Large Transmembrane Protein with EGF-Like
Repeats, Can Antagonize Notch Signaling in Drosophila
Gengqiang Xie1,2,3., Hongtao Zhang1,2., Guiping Du1,2, Qinglei Huang1, Xuehong Liang1, Jun Ma3,4*,
1State Key Laboratory of Brain and Cognitive Science, Institute of Biophysics, the Chinese Academy of Sciences, Beijing, China, 2Graduate School of the Chinese Academy
of Sciences, Beijing, China, 3Division of Biomedical Informatics, Cincinnati Children’s Research Foundation, Cincinnati, Ohio, United States of America, 4Division of
Developmental Biology, Cincinnati Children’s Research Foundation, Cincinnati, Ohio, United States of America
Background: Notch signaling is a highly conserved pathway in multi-cellular organisms ranging from flies to humans. It
controls a variety of developmental processes by stimulating the expression of its target genes in a highly specific manner
both spatially and temporally. The diversity, specificity and sensitivity of the Notch signaling output are regulated at distinct
levels, particularly at the level of ligand-receptor interactions.
Methodology/Principal Findings: Here, we report that the Drosophila gene uninflatable (uif), which encodes a large
transmembrane protein with eighteen EGF-like repeats in its extracellular domain, can antagonize the canonical Notch
signaling pathway. Overexpression of Uif or ectopic expression of a neomorphic form of Uif, Uif*, causes Notch signaling
defects in both the wing and the sensory organ precursors. Further experiments suggest that ectopic expression of Uif*
inhibits Notch signaling in cis and acts at a step that is dependent on the extracellular domain of Notch. Our results suggest
that Uif can alter the accessibility of the Notch extracellular domain to its ligands during Notch activation.
Conclusions/Significance: Our study shows that Uif can modulate Notch activity, illustrating the importance of a delicate
regulation of this signaling pathway for normal patterning.
Citation: Xie G, Zhang H, Du G, Huang Q, Liang X, et al. (2012) Uif, a Large Transmembrane Protein with EGF-Like Repeats, Can Antagonize Notch Signaling in
Drosophila. PLoS ONE 7(4): e36362. doi:10.1371/journal.pone.0036362
Editor: Edward Giniger, National Institutes of Health (NIH), United States of America
Received March 3, 2012; Accepted April 5, 2012; Published April 30, 2012
Copyright: ? 2012 Xie et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported in part by grants from the 973 program (2009CB918702) and the NSFC (31071087) (to RJ) and from NIH and NSF (to JM). The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com (RJ); firstname.lastname@example.org (JM)
. These authors contributed equally to this work.
Notch signaling is an evolutionarily conserved signaling
pathway that regulates a variety of different developmental
processes, including adult homeostasis and stem cell development
[1,2,3,4]. In Drosophila, both the Notch receptor and its canonical
ligands, Delta (Dl) and Serrate (Ser), are transmembrane proteins
with large extracellular domains consisting primarily of EGF-like
repeats. The canonical Notch pathway is activated by an
interaction between the Notch receptor on one cell with its ligand
on the neighboring cell. Such an interaction induces two
consecutive proteolytic processes that result in the release of the
Notch intracellular domain, which is then translocated to the
nucleus and activates transcription of its target genes by interacting
with the DNA-binding protein Suppressor of Hairless (Su(H)) and
the coactivator Mastermind.
Several Notch receptors and a large number of Notch ligands
and co-ligands have been identified in mammals and C. elegans
[5,6]. In Drosophila, a single Notch receptor and two canonical
ligands, Dl and Ser, are well characterized. Recently, an EGF-
repeat-containing protein, Weary (Wry), was identified as a new
Notch ligand important for the maintenance of normal heart
function in the adult fly . The complexity of the biological
processes controlled by the Notch signaling pathway requires
precise regulation of its activity, particularly at the level of ligand-
receptor interactions. For example, the secreted glycoprotein
Scabrous (Sca) has been shown to positively modulate the Notch
activity in regulating proneural development in Drosophila eyes
[8,9]. In addition, Crumbs (Crb), an EGF-like repeat-containing
large transmembrane protein well characterized for its role in
epithelial organization , was recently shown to act as a negative
regulator of Notch signaling in the Drosophila wing . A
significant part of the complexity and specificity of Notch signaling
is derived from the inhibitory action of Notch antagonists.
In this report, we describe the role of a recently identified gene,
uninflatable (uif), in antagonizing Notch signaling activities when
overexpressed. uif was initially characterized for its role in tracheal
development in Drosophila . It encodes a transmembrane
protein with a large extracellular domain consisting of eighteen
EGF-like repeats, a feature common to the Notch receptor and its
ligands. Here, we show that Uif can antagonize the canonical
Notch signaling pathway, acting at a step that is dependent on the
extracellular domain of Notch. Our results suggest a model where
Uif antagonizes Notch activity in a neomorphic manner by
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influencing the accessibility of its extracellular domain available for
interacting with its ligands on neighboring cells during Notch
Ectopic expression of an altered form of Uif causes
phenotypes characteristic of Notch signaling defects
To investigate the role of uif during development, we generated
UAS-Uif transgenic flies that express an altered form of Uif,
referred to as Uif*, which is a nearly full-length protein but has an
altered intracellular domain (see Materials and Methods and
below for details). We assumed initially that Uif* may act in
a dominant negative manner, but further studies made possible by
newly available tools revealed that its biological effects mirror
those of the wild type (wt) Uif protein (see below). Ubiquitous
expression of Uif* caused a semi-lethal phenotype (data not
shown). To circumvent this lethality problem and facilitate the
investigation of the role of uif in development, we used drivers to
express Uif* in a tissue-specific manner. Ectopic expression of Uif*
in the posterior compartment of the wing by engrailed-Gal4 (en-Gal4)
(en-Gal4.Uif*) resulted in significant tissue loss (Figure 1B and
1C). Defects were also observed when Uif* was expressed in other
compartments of the wing. For example, decapentaplegic-Gal4 (dpp-
Gal4) driven expression of Uif* at the anterior-posterior (AP)
boundary of the wing disc caused notched wing in the distal region
of the wing margin (Figure 1D). In addition, expression of Uif*
driven by A9-Gal4 and MS1096-Gal4 in the dorsal compartment of
the wing led to thickened veins (Figure 1E and 1F). These results
show that ectopic expression of Uif* causes patterning defects
The wing phenotypes caused by the ectopic expression of Uif*
are reminiscent of those caused by mutations affecting components
of the Notch signaling pathway, suggesting that ectopically
expressed Uif* may regulate Notch signaling. To evaluate this
possibility, we further targeted Uif* expression in sensory organ
precursor (SOP) cells. Notch signaling is required for SOP
selection and formation [13,14]. Consistent with the wing defects,
Uif* expressed in SOP cells caused SOP selection and formation
defects (Figure 1H and 1I), including patches of bristle loss
(asterisks in Figure 1H, driven by Eq-Gal4) and a nearly complete
loss of bristles in the notum and scutullem (rectangle in Figure 1I,
driven by pannier-Gal4 (pnr-Gal4)). Independent UAS-Uif* trans-
genic lines exhibited similar phenotypes (see Materials and
Methods for details). These results show that ectopic expression
of Uif* in two distinct tissues causes phenotypes that are
characteristic of Notch signaling defects. Since the Uif*-induced
defects in wing patterning and SOP selection were not mitigated
by reducing a wt copy of uif (in uif6/+ heterozygotes; data not
shown), we suggest that ectopically expressed Uif* acts in
a neomorphic manner.
Uif* genetically interacts with Notch pathway
To further investigate the role of Uif* in modulating the Notch
pathway activity, we performed genetic interaction studies
between Uif* and genes encoding Notch signaling components
(Figure 2). A9-Gal4.Uif* adult flies had a weak thickened vein
phenotype (Figure 2B; compare with wt Figure 2A) and N1/+
wings had small notches at the wing margin (arrow in Figure 2C).
However, the combination of N1/+ and A9-Gal4.Uif* led to
a much stronger phenotype, with a severe loss of wing margin
structures and more thickened veins (Figure 2E). Another Notch
allele, N55e11, which on its own only had a very mild wing defect as
heterozygotes (Figure S1A), similarly exhibited genetic interaction
with A9-Gal4.Uif*, leading to enhanced wing phenotypes
(Figure 2F). The thickened vein phenotype of the Dl9P/+ flies
(typically for veins III and V, indicated by arrows in Figure 2D)
was also synergistically enhanced by A9-Gal4.Uif*, with all veins
becoming more broadened and the entire wing becoming smaller
In addition to Notch and Dl, we also analyzed two other
components of the Notch pathway in genetic interaction
experiments. Kuzbanian (Kuz) is a member of the ADAM family
of metalloproteases and mediates S2 cleavage of Notch .
Deltex (Dx) is an E3-ubiquitin ligase, which binds to the
intracellular domain of Notch and positively regulates Notch
signaling . While flies that are heterozygous for Kuz or Dx had
no or mild wing phenotypes on their own (Figure S1B and S1C),
introduction of A9-Gal4.Uif* into these flies led to significantly
enhanced phenotype of thickened veins (Figure 2H and 2I;
compare with Figure 2B for A9-Gal4.Uif* alone). Uif* also
interacted genetically with genes for Notch pathway components
in SOP development. In particular, the neurogenic phenotype of
extra bristles caused by loss of Dl function was potentiated by
a simultaneous expression of Uif* under the control of sca-Gal4
(Figure 2J–2L). Together, these results document a genetic
interaction between Uif* and genes encoding components of the
Notch signaling pathway.
Rescue of Uif*-induced defects by downstream
components of the Notch signaling pathway
Previous studies have identified Notch downstream target genes
that can specifically and selectively suppress phenotypic defects
caused by mutations affecting Notch signaling in different tissues
[17,18]. If Uif* indeed exerts its biological effects by negatively
impacting the Notch signaling pathway, coexpression of the
relevant downstream components of the Notch pathway may
rescue Uif*-induced defects. We tested this idea in both the wing
and the eye. Our results show that the thickened vein phenotype of
A9-Gal4.Uif* adult wings (arrows in Figure 3B) was almost
completely suppressed by A9-Gal4.E(spl)mb (Figure 3D), which
on its own caused slightly thinner veins (Figure 3C and ).
Furthermore, the rough and small eye phenotype of the GMR-
Gal4.Uif* flies (Figure 3F) was significantly alleviated by
coexpression of E(spl)m7 (Figure 3H), which on its own did not
have any detectable abnormality (Figure 3G). These results,
together with those shown in Figure 2, further support the
hypothesis that Uif* perturbs developmental processes through its
inhibitory effects on the canonical Notch signaling pathway.
Expression of Uif* affects the expression of Notch target
Notch signaling controls wing margin formation by activating its
downstream target genes, such as cut, wingless (wg) and vestigial (vg),
in a stripe of cells along the dorsal-ventral (DV) boundary of the
third instar larvae wing imaginal discs [20,21,22,23]. To in-
vestigate at a molecular level the effect of Uif* on Notch signaling,
we analyzed the expression patterns of Notch target genes. Two of
these target genes (Figure 4A and 4C), wg and cut, are known to
respond to low and high thresholds of Notch signaling activity,
respectively . Our results show that, consistently, while Wg
expression was significantly reduced by dpp-Gal4 directed ectopic
expression of Uif* at the AP boundary (Figure 4D, arrow), Cut
expression was completely eliminated (Figure 4B, arrow). In
addition to endogenous target genes of Notch, we also analyzed
two reporter genes that contain Su(H) binding sites, vgBE-lacZ and
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E(spl)mb-lacZ [22,24]. Figure 4F and 4H show that the expression
of both reporters was also significantly decreased at the AP
boundary where dpp-Gal4 expresses (arrows). Together, these
results provide molecular evidence that expression of Uif* directly
affects the activity of the canonical Notch signaling pathway.
Full-length Uif can similarly antagonize Notch signaling
Uif* is almost a full-length form of the protein, with its C-
terminal ten amino acids truncated (see Materials and Methods for
details). It is well documented that removing the intracellular
domains of Dl and Ser can generate dominant negative forms of
these ligands . To determine whether the defects caused by
Uif* might be due to a similar dominant negative effect, we
employed a recently available transgenic fly (GS11655 from the
Kyoto Drosophila Genetic Resource Center) that harbors Gal4-
binding sites upstream of the endogenous wt uif gene. Antibody
staining shows that this endogenous uif gene can respond to the
dpp-Gal4 driver leading to an increased wt Uif level (Figure S2A).
In wing discs of dpp-Gal4.GS11655 flies, Cut protein level was
significantly reduced at the AP boundary (arrow in Figure 5B).
The inhibitory effect of wt Uif on Wg expression was also
detectable (arrow in Figure 5D) but, as expected, weaker than that
on Cut. In addition, we detected notched wings in adults
expressing wt Uif under the control of dpp-Gal4 (asterisk in
Figure 5F). Together, these results show that overexpression of wt
Uif causes molecular and phenotypic defects that are similar to
those of Uif*. However, the effects of Uif* are stronger than wt Uif
(Figure S3), which we attribute to the higher accumulated levels of
Uif* in our experiments (Figure S2 and Discussion). An important
finding here is that these results argue against the possibility that
Uif* acts merely, if at all, as a dominant negative form of the
protein due to its altered intracellular domain, suggesting that
ectopically expressed Uif* and wt Uif are functionally equivalent
(though different in strengths) with respect to the regulation of
Uif* inhibits Notch signaling at a step dependent on the
extracellular domain of Notch
Similar to the Drosophila Notch ligands, Dl, Ser and Wry, Uif
also contains EGF-like repeats. It has been shown that the EGF-
like repeats of the Notch ligands directly interact with the
extracellular domain of Notch . To determine whether Uif
may antagonize Notch signaling in a manner that is dependent on
the extracellular domain of Notch, we compared the effects of Uif*
on Notch receptors that either have or lack this domain.
Expression of full-length Notch (NFL) (driven by dpp-Gal4)
ectopically activated Notch target genes at the AP boundary close
to the DV boundary (arrow in Figure 6A; ). As expected, this
ectopic target gene expression was significantly suppressed by
coexpression of Uif* (Figure 6B). However, Uif* had no effect on
Figure 1. Ectopic expression of Uif* causes phenotypes that are characteristic of Notch signaling defects. (A) A wt adult wing. (B and C)
Targeted Uif* expression under the control of en-Gal4 causes loss of wing margin structures in the posterior wing compartment. These two panels
show different expressivity, ranging from a partial loss of wing margin (arrow in B) to an almost complete loss of the posterior wing margin (black line
in C). (D) A dpp-Gal4.Uif* adult wing shows wing margin loss (arrow) at the most distal tip area of the wing and an occasional loss of the anterior
cross vein (arrowhead). (E and F) Thickened veins, which resemble an aspect of the Notch loss of function phenotypes (particularly veins III and V,
arrows), observed in adult wings of A9-Gal4.Uif* (E) and MS1096-Gal4.Uif* (F) flies. (G–I) Expression of Uif* in the notal region causes losses of
sensory bristles. (G) A wt adult notum with a regular pattern of sensory bristles. (H) The notum of Eq-Gal4.Uif* flies shows random losses of
microchaeta (asterisks). (I) Expression of Uif* in the notum controlled by pnr-Gal4 leads to a great loss of sensory bristles (rectangle).
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a constitutively active form of Notch that lacks its extracellular
domain (NECN) and activated its downstream target genes in
a ligand-independent manner (compare Figure 6D with 6C).
These results suggest that Uif modulates Notch signaling at a step
that is dependent on the extracellular domain of Notch.
Uif* inhibits Notch signaling through a cis mechanism
The ligands Dl and Ser can regulate Notch signaling through
[20,21,27,28,29,30,31,32,33]. Paracrine interaction leads to Notch
activation in trans (referred to as trans activation) whereas autocrine
interaction leads to Notch inhibition in cis (referred to as cis
inhibition). Both effects are achieved through physical interactions
between the extracellular domains of Notch and its ligands .
To further clarify the nature of Uif* action with regard to its
topological relationship with Notch, we took advantage of an
ectopic expression system, where both cis inhibitory and trans
activating effects of Notch ligands are exhibited simultaneously
[34,35]. Here, we used dpp-Gal4 to drive Ser or Dl ectopic
expression in the wing imaginal discs (Figure 7). In addition to trans
activation exhibited by the ectopic Wg expression, the cis
inhibitory effects of these ligands were simultaneously exhibited
by a reduction of Wg expression levels within the domain of
ligand-expressing cells (marked by GFP; asterisk in Figure 7A and
arrow in Figure 7C). However, such cis inhibitory effects are
incomplete and detectable only in cells expressing ligands at high
levels. Coexpression of Uif* with the ligands greatly enhanced the
cis inhibition, leading to a complete elimination of Wg expression
in almost all ligand-expressing cells (arrow in Figure 7B and
asterisk in 7D). These results suggest that ectopic expression of
Uif* negatively regulates Notch signaling through a cis inhibitory
mechanism, either working on its own or, more likely (as in our
experimental setting), working in concert with Ser or Dl.
The canonical Notch signaling pathway is one of a limited
group of pathway modules that transduce signals from outside the
cell to alter gene expression inside the nucleus [1,2,3]. These
Figure 2. Uif* genetically interacts with genes for the Notch signaling pathway. (A) A wt adult wing. (B) An adult wing of the A9-Gal4.Uif*
fly showing mild thickened vein phenotype that resembles Dl loss of function phenotype (D). (C) A N1/+ wing showing a typical small notch at the
distal region of the wing margin (arrow). (D) A Dl9P/+ wing showing the thickened vein phenotype, particularly in the distal region of veins II and V
(arrows). Wings of either N55e11/+, kuze29-4/+, or Dx1/+ adult flies have no or mild defects (Figure S1). (E–I) Wings of A9-Gal4.Uif* in combination with
one copy of mutation of different Notch pathway components showing enhanced phenotypes as compared with either of them alone. Very small
wings with a great loss of wing margin structures and thickened veins are shown in A9-Gal4.UAS-Uif*; N1/+ (E) and A9-Gal4.UAS-Uif*; N55e11/+ (F);
blistering wing phenotype is also observed in the majority of adult flies (see Discussion). Thickened veins are shown in A9-Gal4.UAS-Uif*; Dl9P/+ (G)
A9-Gal4.UAS-Uif*; kuze29-4/+ (H) and A9-Gal4.UAS-Uif*; Dx1/+ (I) as compared with A9-Gal4.UAS-Uif* (B). (J–L) The notal region of an adult fly
expresses UAS-Uif* (J), UAS-DlDN(K) or UAS-DlDNplus UAS-Uif* (L) under the control of sca-Gal4. The neurogenic phenotype of extra bristles caused by
the loss of Dl function is potentiated by the simultaneous expression of Uif*.
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pathways together orchestrate the developmental processes that
can be dauntingly complex. Yet it is the same modules that are
used repeatedly, not only in different organisms, but also in vastly
different processes within an organism [4,36]. Thus, how these
pathway modules are activated in a specific manner, with regard
to not only space and time but also the quantity of their signaling
output, represents a fundamental question in developmental
biology. Here we describe a newly characterized protein, Uif,
which can antagonize the canonical Notch signaling pathway in
a neomorphic manner. These findings underscore the importance
of the precise tuning of Notch activity in normal patterning.
EGF-like repeats are a common feature of Notch receptors,
ligands and co-ligands [6,37]. While Uif was originally character-
ized for its role in tracheal development, its EGF-like repeats
suggest a possible role in Notch signaling. Our results are
consistent with a model where ectopically expressed Uif may
modulate the accessibility of the extracellular domain of Notch to
its ligands during activation. It is possible that the EGF-like repeats
of Uif directly interact with the extracellular domain of Notch to
exert its inhibitory effect in a manner similar to the cis inhibition
by Notch ligands themselves [20,28,29,30,31,32,33]. Our finding
that Uif* acts on Notch through a cis inhibitory mechanism
(Figure 7) is supportive of this possibility. In our experiments, Uif*
is more effective than wt Uif in antagonizing Notch, and this
difference may be attributed to the difference in their expression
levels (Figure S2). These results suggest that ectopically expressed
Uif* and wt Uif have a similar neomorphic function in regulating
A proposed neomorphic function of Uif* and Uif in Notch
signaling is consistent with our results of loss of function analysis of
uif. Knockdown (assayed for adult wing phenotypes and Notch
target gene expression using independent RNAi lines; data not
Figure 3. Expression of Notch target genes rescues Uif*-induced defects. (A) A wt wing. Expression of Uif* under the control of A9-Gal4
causes thickened vein phenotype with broadened veins III and V (arrows in B). This defect can be significantly alleviated by coexpression of a Notch
downstream component, E(spl)mb (arrows in D). (C) shows control wing of A9-Gal4.E(spl)mb flies. A small and rough eye phenotype (F) in GMR-
Gal4.Uif* flies is significantly rescued by coexpression of E(spl)m7 (H). (E) and (G) show control eyes of GMR-Gal4/+ and GMR-Gal4.E(spl)m7 flies,
Figure 4. Uif* reduces the expression of Notch target genes.
Expression of Notch target genes, Cut (A and B), Wg (C and D), vgBE-lacZ
(E and F) and E(spl)mb–lacZ (G and H), in the third instar wing discs of
wild type larvae, with (B, D, F and H) or without (A, C, E and G) Uif*
overexpression. Genotypes are: (A and C) dpp-Gal4 UAS-GFP/+; (B and D)
dpp-Gal4 UAS-GFP/UAS-Uif*; (E) dpp-Gal4 UAS-GFP/vgBE-lacZ; (F) dpp-
Gal4 UAS-GFP/vgBE-lacZ UAS-Uif*; (G) E(spl)mb–lacZ/+; dpp-Gal4 UAS-
GFP/+ and (H) E(spl)mb–lacZ/+; dpp-Gal4 UAS-GFP/UAS-Uif*. Arrows
indicate a loss or a decreased expression of the Notch target genes at
the AP boundary of the wing discs where Uif* was expressed under the
control of dpp-Gal4 (B, D, F and H).
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shown) or knockout (assayed for Notch target gene expression in
somatic mutant clones; Figure S4) of uif revealed neither Notch
loss of function nor gain of function phenotypes. However, it
remains formally possible that the endogenous uif gene has a native
role in regulating Notch signaling in tissues or cells (other than
those that we have examined) at a time during Drosophila
development. Further studies are required to investigate this
Figure 5. Full-length wt Uif antagonizes Notch signaling. Overexpression of wt Uif by dpp-Gal4.GS11655 leads to a significant reduction in
the levels of both Cut and, to a lesser degree, Wg. (A) and (C) show Cut and Wg expression patterns in wing discs from the dpp-Gal4/+ control flies,
respectively. (B) and (D) show the Cut and Wg levels in wing discs from dpp-Gal4.GS11655 flies, respectively (see regions pointed by arrows). GFP in
(A9–D9) shows the expression pattern of dpp-Gal4. A notched wing detected in a dpp-Gal4.GS11655 adult fly (F), compared with a dpp-Gal4/+ control
wing (E). Flies were reared at 29uC.
Figure 6. The inhibitory effect of Uif* is dependent on the extracellular domain of Notch. Ectopic expression of the full-length Notch (NFL)
under the control of dpp-Gal4 induces aberrant Wg (red) expression at the AP boundary where it intersects with the DV boundary (white arrow in A)
(A and A9). GFP (green) marks dpp-Gal4 positive cells (A9, B9, C9 and D9). Coexpression of Uif* with NFLreduces the ectopic induction of Notch
signaling mediated by NFLat the intersection between AP and DV boundaries (white arrow in B) (B and B9). Ectopic expression of the membrane
tethered active version of Notch (NECN) induces Wg (red) expression in the dpp-Gal4 region that is marked by GFP (green) (C and C9). Coexpression of
Uif* does not alter the Wg expression that is induced by NECN(D and D9).
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The biological activities of Uif are not restricted to regulating
role in tracheal inflation underscores the complexity of its biological
activities. In addition to the EGF-like repeats, Uif also contains
These domainsincludea C-type lectin-like (CLECT) domain, three
CUB domains, eight complement control protein (CCP) domains,
two coagulation factor 5/8 C-terminal (FA58C) domains and three
hyaline repeat (HYR) domains. Both CLECT and FA58C domains
are putative carbohydrate binding domains known to play
important roles in many diverse processes [38,39]. The CUB
domain is an evolutionary conserved protein domain found almost
exclusively in extracellular and plasma membrane-associated
proteins . HYR is an immunoglobulin fold domain likely
Sushi domains or Short Consensus Repeats (SCR), exist in a wide
variety of complement and adhesion proteins . These domains
suggest that Uif may also play a role in cell adhesion. Indeed, in
a recent genetic modifier screen, uif was identified as a regulator
(Mod29) of the Drosophila Dystroglycan-Dystrophin Complex,
a specialized celladhesion complex. Mod29/Uif wassuggested
to play roles in multiple developmental processes, including wing
vein formation, muscle and photoreceptor axon development, and
oogenesis . Although it remains to be investigated whether Uif,
a large regulator with multiple conserved protein domains, may
functionally connect distinct cellular processes, our own unpub-
lished data offer some speculative insights. In particular, the
blistering wing phenotype caused by knockdown of Dl or Ser 
can be fully rescued by depletion of uif (data not shown), suggesting
that Uif may functionally extend the role of Notch ligands to cell
adhesion. UifisanN-glycosylated protein,amodificationsharedby
complexes . Understanding the full spectrum of the biological
functions of Uif during development and, importantly, its potential
role in harmonizing different cellular processes, represents future
Materials and Methods
Generation of UAS-Uif* and UAS-UifRNAitransgenic flies
A pUAST-Uif* construct was made by inserting a part of the
uif cDNA sequence that encodes the first 165 amino acids of Uif
and a genomic DNA fragment encoding the remaining amino
acids of Uif-PA into the pUAST vector. This transgene is
expected to encode a protein that lacks the last ten amino acids
at the C-terminus of the predicted full-length Uif protein (amino
acid 3548 to amino acid 3557), with two amino acid changes
(N1567D and A3134T) and an addition of three extra amino
acids (SGR) immediately after amino acid 165 resulting from the
insertion of a restriction enzyme Not I site in the coding
sequence. After standard P element-mediated germline trans-
formation, three independent lines of transgenic flies that carry
pUAST-Uif* were obtained, all of which resulted in similar
phenotypes when expressed under different Gal4 drivers tested.
Immunostaining with antibodies against extracellular and in-
tracellular domains of Uif demonstrated that Uif* is properly and
stably expressed under the control of dpp-Gal4 (Figure S2B and
data not shown).
To construct UAS-UifRNAiflies, two pieces of non-overlapping uif
coding sequence were cloned into the pWIZ vector . Germline
transformants that carry each sequence were generated by
standard procedures at the Rainbow Transgenic Flies Inc
(Camarillo, CA). At least three independent lines for each RNAi
constructs were tested for the RNAi strength and specificity.
Null mutant of uif and other Drosophila strains
uif null mutants were generated by homologous recombination
mediated gene targeting strategy [47,48]. One of the alleles,
designated uif6, which was molecularly verified and can be
Figure 7. Uif* enhances cis inhibition of Notch signaling by its ligands. (A and A9) dpp-Gal4.UAS-Ser leads to both cis inhibition (in the inner
region of the dpp-Gal4 expressing, GFP+domain in the ventral part of the disc; marked by the asterisk) and trans activation of Wg (in cells
neighboring to the dpp-Gal4 expressing domain in the ventral compartment of wing disc; marked by arrowheads). The cis inhibition is incomplete
and, thus, Wg expression (arrows) is detected in the outer region of the dpp-Gal4 expressing domain. Coexpession of Uif* enhances cis inhibition,
leading to Wg reduction inside the dpp-Gal4 expressing domain, without affecting trans activation (arrowheads in B and B9). Expression of Dl by dpp-
Gal4 causes Wg expression mainly in the dorsal compartment both inside and outside of the dpp-Gal4 regions (C). Wg protein level inside of the dpp-
Gal4 expressing domain is lower, reflective of cis inhibition (arrow in C). When Uif* is coexpressed, this cis inhibition is enhanced, leading to a nearly
complete loss of Wg expression inside of the dpp-Gal4 expression domains (asterisks in D and D9), without affecting trans activation (outside of dpp-
Gal4 expression domain; arrowheads in D and D9). GFP (green) marks the domain where dpp-Gal4 is expressed (A9, B9, C9 and D9). See the main text
for further details.
Uif Can Antagonize Notch Signaling
PLoS ONE | www.plosone.org7 April 2012 | Volume 7 | Issue 4 | e36362
completely rescued by a genomic transgene of uif (Figure S4 and
data not shown), was used in this study. The transgenic fly strain
used for genomic rescue was generated by direct injection of
a BAC clone (CH321-83F13, from P[acman] BAC libraries,
BPRC (BACPAC Resources Center)) that contains the uif
genomic fragment into flies, which harbor both the vas-phiC31
transgene (on X chromosome) and a attP target site (on 3rd
chromosome) . Other fly strains that were used in this study
include: w1118, UAS-Uif* (this paper), Eq-Gal4 , pnr-Gal4,
Dl9P/TM3 Sb, N1, N55e11, kuze29-4, Dx1, sca-Gal4, UAS-DlDN, y1
Kyoto), en-Gal4, GMR-Gal4, UAS-GFP, dpp-Gal4, MS1096-Gal4,
A9-Gal4, vgBE-lacZ , E(spl)mb-lacZ, y w hsFlp122; ubi-GFP
FRT40A/CyO, UAS-UifRNAi-1, UAS-UifRNAi-2, UAS-E(spl)mb, UAS-
E(spl)m7, UAS-NFL, UAS-NECN, UAS-Dl30 and UAS-Ser.
All flies were from the Bloomington Drosophila Stock Center at
Indiana University unless otherwise stated. All crosses were
carried out at 25uC according to standard procedures unless
Generation of anti-Uif antibodies
We generated antibodies against the extracellular domain and
the intracellular domain of Uif. Briefly, uif coding sequences for
amino acids 1113–1343 (extracellular domain) and 3440–3548
(intracellular domain) were cloned into the pET21b(+) vector.
The proteins were expressed in BL21 E. coli cells and purified
according to Qiagen Ni-NTA handbook. Purified proteins were
used to generate antibodies in rabbits at the Cocalico Biologicals
Inc (Reamstown, PA). The anti-Uif sera were subsequently
affinity purified with protein G beads (Invitrogen) prior to use in
Immunostaining of wing imaginal discs was performed as
previously described [52,53]. In addition to antibodies against Uif
(see above), the following primary antibodies were used: mouse
anti-Wg (4D4, 1:20, the Developmental Studies Hybridoma Bank
[DSHB], University of Iowa, Iowa City, IA, USA), mouse anti-Cut
(2B10, 1:20, DSHB), rabbit anti-GFP (1:1000, Invitrogen) and
rabbit anti-b-Galactosidase (1:1000; Sigma). The secondary
antibodies used were conjugated to FITC or Cy3 (Jackson
Immunoresearch), each diluted at 1:200. Images were captured
on a Leica TSC SP5 confocal laser scanning microscope and
processed using Adobe Photoshop.
heterozygous flies. (A) An adult wing of N55e11/+ flies shows
a mild delta vein phenotype in the most distal regions of veins IV
Adult wings of N55e11/+ +, kuze29-4/+ and Dx1/+
and V (arrows; compare with a wt wing in Figure 1A). (B and C)
kuze29-4/+ and Dx1/+ adult wings have normal wing pattern.
different levels in the wing disc. Wing discs immunnostained
with anti-Uif antibody showing the ectopic expressing level of wt
Uif (A and A0) or Uif* (B and B0). GFP marks the dpp-Gal4
expressing cells in A9, A0, B9 and B0. All experiments shown here
were performed side by side with images captured and processed
under identical settings. Flies were reared at 18uC.
Wild type Uif and Uif* are expressed at
on Cut expression. (A) Cut expression at the DV boundary in
the control wing disc (dpp-Gal4/+). Expression of wt Uif by dpp-
Gal4.GS11655 causes a detectable reduction of the Cut level at
the AP boundary (arrow in B). Panel C shows a stronger reduction
of Cut expression caused by Uif* (arrow). GFP marks dpp-Gal4
expressing cells. All experiments shown here were performed side
by side with images captured and processed under identical
Comparison of the effects of wt Uif and Uif*
in uif mutant clones. (A) Cut expression pattern in the wing
disc with FRT40A mock clones, marked by the absence of GFP
(A9). (B) No detectable changes of Cut expression pattern in the uif6
mutant clones (marked by GFP negative cells in B9) comparing
with the mock clones. (A0 and B0) are the overlaid images. (D)
Adult wing with uif6mutant clones show wrinkles and reduced size
as compared with wild type (C), which is fully rescued by a copy of
uif genomic DNA (E).
Notch signaling is not detectably upregulated
We thank Dr. Hugo J. Bellen, Dr. Cheng-Ting Chien, Dr. Hiroshi Nakato,
Dr. Konrad Basler, the Vienna Drosophila RNAi Center, the Kyoto
Drosophila Genetic Resource Center, the Bloomington Drosophila Stock
Center and the Developmental Studies Hybridoma Bank, Iowa, for
reagents. We thank members of the Jiao lab and Dr. Li Liu’s lab at IBP for
stimulating discussions, David Cheung of CCHMC for assistance
throughout the work, and Dr. Junbo Liu and Dr. Jarek Meller (both of
CCHMC) for discussions and assistance during the initial phase of the
work. We are grateful to the anonymous reviewer and the PLoS One editor
for constructive suggestions.
Conceived and designed the experiments: RJ JM GX HZ. Performed the
experiments: GX HZ GD QH. Analyzed the data: RJ JM GX HZ.
Contributed reagents/materials/analysis tools: XL. Wrote the paper: GX
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PLoS ONE | www.plosone.org9 April 2012 | Volume 7 | Issue 4 | e36362