Ligand-binding and signaling properties of the Ax[M1] form of Notch

Lidia Pereza, Marco Milanb, Sarah Brayc, Stephen M. Cohena,*

aEuropean Molecular Biology Laboratory, Meyerhofstr 1, 69117 Heidelberg, GermanybIcrea and Parc Cientific de Barcelona, Josep Samitier, 1-5, 08028 Barcelona, Spain

cDepartment of Anatomy, Cambridge University, Cambridge, UK

Received 30 June 2004; received in revised form 18 November 2004; accepted 28 November 2004

Available online 30 January 2005

Abstract

The Abruptex class of Notch alleles has attracted interest because they exhibit some properties that are best explained in terms of increased

activity and others that are best explained in terms of reduced activity in vivo. Here, we report a comparison of the properties of

Abruptex[M1] and wild-type Notch as ligand binding receptors. Abruptex[M1] showed less activity than wild-type Notch in its ability to bind

Delta and Serrate and was expressed at reduced levels on the cell surface. When differences in expression level were taken into account,

Abruptex[M1] was comparable to Notch in its sensitivity to ligand-induced activation of reporter gene expression. Abruptex[M1] was also

comparable to Notch in its requirement for modification by Fringe and in being sensitive to cis-dowregulation by co-expressed ligands. By

the available criteria Abruptex[M1] exhibits less activity than Notch. To explain the ectopic activity of Abruptex[M1] in vivo we suggest that

it may be necessary to invoke an altered response to an as yet unidentified ligand or cofactor.

q 2005 Elsevier Ireland Ltd. All rights reserved.

Keywords: Notch; Signal transduction; Pattern formation

1. Introduction

Notch signaling is involved in a variety of cell fate

decisions during development. In the wing imaginal disc

Notch is activated at the boundary between dorsal and

ventral compartments, where it induces the expression of

vestigial, cut and Wingless (Diaz-Benjumea and Cohen,

1995; Rulifson and Blair, 1995; de Celis et al., 1996;

Doherty et al., 1996; Kim et al., 1995; Neumann and Cohen,

1996). The combined activity of Notch and Wingless

organizes growth and patterning of the wing disc (Baonza

and Garcia-Bellido, 1999; Giraldez and Cohen, 2003;

Johnston and Sanders, 2003). One of the most intriguing

features of the Notch signaling system in this context is the

diversity and complexity of the mechanisms that are used to

limit Notch activity to the DV boundary. Notch is expressed

in all cells of the imaginal discs. Its ligands, Delta and

0925-4773/$ - see front matter q 2005 Elsevier Ireland Ltd. All rights reserved.

doi:10.1016/j.mod.2004.12.007

* Corresponding author. Tel.: C49 6221 387 172; fax: C49 6221 387

166.

E-mail addresses: cohen@embl.de (S.M. Cohen), scohen@embl-

heidelberg.de (S.M. Cohen).

Serrate, are also broadly expressed in the wing disc, yet

Notch activity is limited to a narrow stripe of cells along the

DV boundary (Diaz-Benjumea and Cohen, 1995; Rulifson

and Blair, 1995; de Celis et al., 1996; Doherty et al., 1996;

Kim et al., 1995). Four distinct mechanisms have been

implicated in limiting Notch activity to cells immediately

adjacent to the DV boundary.

The first mechanism depends on modification of Notch

by the glycosyltransferase enzyme Fringe (Moloney et al.,

2000; Bruckner et al., 2000). Fringe acts as a glycosyl-

transferase enzyme to add N-Acetyl-Glucosamine to O-

linked Fucose residues on the EGF modules of the

extracellular domains of Notch. O-linked Fucose is required

for Notch activation (Okajima and Irvine, 2002), and

extension of the sugar chain by Fringe affects the selectivity

of ligand binding by Notch (Bruckner et al., 2000; Chen

et al., 2001; Hicks et al., 2000; Moloney et al., 2000;

Okajima et al., 2003). Fringe and the Notch-activating

ligand Serrate are both expressed in dorsal cells under

control of Apterous (Diaz-Benjumea and Cohen, 1993;

Blair et al., 1994; Irvine and Wieschaus, 1994). Modifi-

cation by Fringe renders Notch insensitive to activation by

Mechanisms of Development 122 (2005) 479–486

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L. Perez et al. / Mechanisms of Development 122 (2005) 479–486480

Serrate (Panin et al., 1997; Fleming et al., 1997; Johnston et

al., 1997). Consequently, Serrate only activates Notch in

adjacent ventral compartment cells in which Notch has not

been modified. Conversely, despite being coexpressed with

Notch in all ventral cells, Delta only activates Notch in

dorsal cells (de Celis et al., 1996; Doherty et al., 1996).

In vitro binding assays have shown that modification by

Fringe increases the affinity of Notch for Delta (Bruckner

et al., 2000; Okajima et al., 2003). These findings therefore

suggest that the level of Delta expression in the V

compartment is too low to activate unmodified Notch, so

that Delta only activates Notch in adjacent dorsal cells in

which Notch has been modified by Fringe. Asymmetric

signaling by Serrate and Delta limits Notch activation to

cells near the DV boundary; however, additional mechan-

isms are needed to constrain Notch activity to a stripe of

cells immediately adjacent to the boundary.

A second important mechanism for limiting Notch

activation involves a relay of Notch signaling through its

target gene Wingless. Notch activation induces Wingless

expression in cells at the DV boundary. High levels of

Wingless signaling in turn induces expression of the two

Notch ligands, Serrate and Delta in adjacent cells, so that the

boundary is flanked by cells expressing the two ligands. The

elevated levels of ligand expressed in these flanking cells

serve to repress Notch signaling in these cells (de Celis and

Bray, 1997; Micchelli et al., 1997). Coexpressed ligands

interact with Notch and because these complexes are not

found at the cell surface, may reduce the level of Notch

available for activation by ligand at the cell surface

(Sakamoto et al., 2002). This conclusion is based on the

observations that Notch target genes are ectopically

expressed in clones of cells mutant for the ligands and

that cells ectopically expressing the ligands induce Notch

targets only in adjacent cells, but not in the ligand

expressing cells themselves. Elevated expression of Notch

can overcome the inhibitory effects of the ligands (Doherty

et al., 1996; Klein et al., 1997), suggesting that inhibition

involves a process of titration through binding between the

ligand and the receptor in cis.

The first and second mechanisms reflect the regulation of

Notch activity at the DV boundary of the wing disc. The

third and fourth mechanisms appear to be overlaid on top of

these context-specific mechanisms. Dishevelled is known as

a mediator of Wg signaling (Klingensmith et al., 1994;

Noordermeer et al., 1994). In addition, Dishevelled has been

shown to bind to the intracellular domain of Notch and to

limit its signaling activity so that the domain in which Notch

can induce wing margin formation is expanded in clones of

dishevelled mutant cells (Rulifson et al., 1996; Axelrod

et al., 1996). The POU-homeodomain protein Nubbin is

expressed throughout the wing pouch where it limits the

ability of Notch to activate target genes, including wingless

and vestigial, presumably by acting as a repressor in

opposition to transcriptional activation by the Notch

Suppressor of Hairless complex (Neumann and Cohen,

1998).

The prospect of gaining further insight into the regulation

of Notch activity has been offered by point mutations that

alter Notch activity. The exodomain of Notch contains 36

EGF modules. EGF modules 11 and 12 have been

implicated in ligand binding and at least one loss of function

allele of Notch affects repeat 11 (Rebay et al., 1991; de Celis

et al., 1993; Lieber et al., 1992). Other loss of function

alleles affect other EGF modules or the intracellular domain

of the protein. Two gain-of-function alleles increase Notch

activity in a ligand independent manner and appear to affect

the third lin12/Notch repeat (Brennan et al., 1997). Notch

undergoes a series of constitutive and ligand induced

processing steps to generate an active receptor (reviewed

in Fortini, 2002; Baron, 2003; Selkoe and Kopan, 2003).

These alleles may affect Notch processing to render a mildly

constitutively active protein. The Abruptex class of alleles is

more complex and appears to have both gain of function and

loss of function qualities (Heitzler and Simpson, 1993;

Brennan et al., 1997; de Celis and Garcia-Bellido, 1994).

These alleles increase Notch activity in the wing disc, but do

not simply activate the protein in a ligand independent

manner. In this report we make use of several direct

measures of the function of Notch as a receptor protein to

examine the basis for the gain of function properties of an

Abruptex allele. Ax[M1] is due to a change of Cysteine 999

to Tyrosine in EGF module 25 (Brennan et al., 1997), which

is expected to alter the structure of the EGF module, yet it

does not produce a simple loss of Notch function. Although

in vivo this mutation results in phenotypes that equate with

ectopic Notch activity, by all of the criteria examined here

Ax[M1] shows less activity than the wild-type form of the

Notch protein. We conclude that the reason for the ectopic

activity of Notch in this allele cannot be explained solely in

terms of its effects on the function of Notch as a receptor for

Delta or Serrate or by its refractoriness to cis-down-

regulation by its ligands.

2. Results

The Ax[M1] mutation was selected for this study because

it has been shown to cause ectopic activation of Notch target

gene expression in the wing disc (de Celis and Bray, 2000;

Ju et al., 2000; Fig. 1). The Notch targets Wingless and Cut

are normally expressed in a narrow band of cells on each

side of the dorso–ventral compartment boundary in wild-

type discs. These domains are expanded in both compart-

ments of the Ax[M1] mutant disc (Fig. 1B,C). The excess

activity of Ax[M1] appears to be ligand dependent (1) it is

enhanced by increasing the dosage of Delta (de Celis and

Garcia-Bellido, 1994) and (2) ectopic activity is not

observed in all cells but tends to be concentrated close to

the DV boundary (though in some discs the ectopic domains

can extend far from the boundary, arrow Fig. 1B).

Fig. 1. Comparison of Notch and Ax[M1] activity in the wing disc. (A, B) Wingless protein expression in wild-type (A) and Ax[M1] hemizygous third instar

wing imaginal discs (B). Arrow in (B) points to a broad domain of ectopic Wg expression in the lateral part of the dorsal compartment. (C) Ax[M1] hemizygous

third instar wing imaginal disc labeled with antibody to Cut protein (red). The dorsal compartment is labeled by expression of an apterous reporter gene

(green). Note that cut is ectopically expressed in both dorsal and ventral cells. The degree of Wg and Cut misexpression is variable. The examples presented

here show an intermediate severity (for examples of stronger misexpression see de Celis and Bray, 2000).

Fig. 2. In vitro binding of Notch-AP and Ax[M1]-AP fusion proteins.

Notch-AP and Ax[M1]-AP were transfected into S2 cells with or without

Fringe. The secreted fusion proteins were recovered in conditioned

medium. The concentration of the Notch-AP and Ax[M1]-AP fusion

proteins was normalized by diluting with culture medium. Conditioned

media were incubated for 90 min with cells transfected to express Delta,

Serrate or with control S2 cells, washed and bound AP activity was

measured as described (Bruckner et al., 2000). The data are the average of

four replicatesGSD.

L. Perez et al. / Mechanisms of Development 122 (2005) 479–486 481

Fringe activity makes Notch insensitive to Serrate, with

which it is coexpressed in dorsal cells, and more sensitive to

Delta signaling from ventral cells (Panin et al., 1997;

Fleming et al., 1997; Johnston et al., 1997). A priori, the

ectopic activity of Ax[M1] could be due to enhanced

sensitivity to Serrate (i.e. loss of Fringe-induced insensitiv-

ity to Serrate) or due to increased sensitivity to Delta.

To compare the activities of Notch and Ax[M1] more

directly we have turned to in vitro ligand binding assays

(Bruckner et al., 2000). Secreted alkaline phosphatase (AP)

fusion proteins were prepared with the extracellular

domains of wild-type Notch and the Ax[M1] mutant form

of Notch. These proteins were expressed in S2 cells or in S2

cells cotransfected to express Fringe. For use in binding

assays the concentration of the Notch-AP and Ax[M1]-AP

fusion proteins was normalized by adjusting the conditioned

media to equivalent levels of alkaline phosphatase activity.

The conditioned media were then incubated with control S2

cells or with S2 cells transfected to express Delta or Serrate

and the amount of AP fusion protein bound to the cells was

measured (Fig. 2). There was no significant difference in

binding of Notch-AP and Ax[M1]-AP to control S2 cells.

Notch-AP produced in S2 cells bound detectably to Serrate-

expressing cells at 2-fold above background levels. Binding

of Notch-AP produced in Fringe-expressing cells was not

distinguishable from background, indicating that modifi-

cation of Notch by Fringe reduces binding to Serrate.

Although the magnitude of Notch-AP binding to Serrate

expressing cells in vitro was low, the effect of Fringe

modification is consistent with its effects in vivo. Interest-

ingly, binding of unmodified Ax[M1]-AP to Serrate

expressing cells was not distinguishable from background

levels and there was no detectable difference when Ax[M1]

was modified by Fringe. This indicates that the Ax[M1]

protein does not bind to Serrate better than wild-type Notch

and suggests that ectopic activation of Ax[M1] in the dorsal

compartment is unlikely to be due to increased affinity for

Serrate.

We next asked whether Ax[M1] activity could be

explained by increased affinity for Delta. As reported

previously (Bruckner et al., 2000), Notch-AP produced in

Fringe-expressing S2 cells bound considerably better to

cells expressing Delta than unmodified Notch-AP produced

in S2 cells (Fig. 2). Ax[M1]-AP produced in Fringe-

expressing cells bound to cells expressing Delta at

approximately 1/10th the level of Notch-AP (note that

input AP levels are equal). Binding of unmodified Ax[M1]-

AP produced in S2 cells was not distinguishable from

background. These observations indicate first that Ax[M1]

binding to Delta is regulated by Fringe, in a manner similar

to wild-type Notch. Second, they show that Fringe-modified

Ax[M1] binds to Delta less well than to comparably

prepared Notch. These observations suggest that ectopic

activation of Ax[M1] cannot be explained by increased

affinity for Delta. Nor can it be explained by insensitivity of

Ax[M1] to modification by Fringe.

The preceding experiments suggest that the ectopic

activation of the Ax[M1] mutant protein cannot be

L. Perez et al. / Mechanisms of Development 122 (2005) 479–486482

explained in terms of its intrinsic ability to bind to Serrate or

Delta. We next considered the possibility that Ax[M1]

might differ from Notch in its ability to function as a

receptor at the cell surface. To address this we compared the

binding of a secreted Delta-AP fusion protein to S2 cells

expressing full-length Notch or Ax[M1] as transmembrane

proteins. Cells were co-transfected with myc-tagged Fringe

or with a myc-tagged mutant form of Fringe that lacks

catalytic activity (Bruckner et al., 2000). Delta-AP binding

was not distinguishable from background for cells expres-

sing unmodified Notch or Ax[M1]. Delta-AP bound

considerably better to Fringe-modified Notch than to

Fringe-modified Ax[M1], although the level of expression

of the two forms of Notch was comparable (Fig. 3A). As in

the preceding experiments, Fringe-modified Ax[M1]

showed a reduced ability to bind Delta compared to

Fringe-modified wild-type Notch.

To examine the basis for this difference in more detail,

we prepared versions of Notch and Ax[M1] with an HA

epitope-tag in the extracellular domain and verified that they

bound Delta comparably to the untagged proteins (not

shown). Antibody to HA was used to surface label cells

transfected to express Notch-HA and Ax[M1]-HA and

Fig. 3. Reduced binding of Delta to Ax[M1]. (A) In vitro binding of a secreted De

Notch or Ax[M1]. Light grey bars indicate cells cotransfected to express myc

catalytically inactive form of myc-tagged Fringe. The data are the average of four

specific background. Background was subtracted from the Notch and Ax[M1] bind

Ax[M1]. Immunoblots of the cell lysates are shown below probed with anti-Notch

Notch is not expressed at detectable levels in the control S2 cells, so the western s

(B) HA-tagged versions of Notch and Ax[M1] were cotransfected with Fringe in S

anti-HA for 90 min followed by binding of AP conjugated anti-rat IgG for 90

transfected to express Fringe were used to determine the level of non-specific

background subtractionGSE. An immunoblot of the cell lysates probed to detect N

probed together, but were not in adjacent lanes (the intervening lanes have been

bound antibody was detected with AP-conjugated second-

ary antibody. Cells transfected with the untagged proteins

were used to determine the level of non-specific background

binding of the antibodies. Fig. 3B shows that the relative

level of cell surface expression of Notch-HA was 3–4-fold

higher than Ax[M1]-HA after background subtraction. The

reduced level of cell surface expression of Ax[M1]-HA can

therefore account for much of the difference in the ability of

Ax[M1]-HA and Notch to bind Delta-AP.

We next compared the ability of Delta to signal via Notch

and Ax[M1] and activate transcription of a Notch-

dependent reporter gene. Cells were transfected to express

Notch or Ax[M1] and Fringe together with a Notch-

responsive firefly luciferase reporter construct containing

six Suppressor of Hairless binding sites (three copies of the

paired Su(H) binding sites from E(spl)m8) and a control

renilla luciferase plasmid to control for transfection

efficiency. These cells were co-cultured with Delta expres-

sing cells to provide the activating ligand in trans or with S2

cells to control for ligand-independent activation (Fig. 4,

values normalized for transfection efficiency). For cells

transfected to express Notch, co-culture with Delta cells

caused a 10-fold induction of reporter gene expression over

lta-AP fusion protein to S2 cells (control) or S2 cells transfected to express

-tagged Fringe. Dark grey bars indicate cell cotransfected to express a

replicatesGSD. The level of Delta-AP binding to S2 cells provides the non-

ing data to derive the normalized value of 5.5-fold reduced Delta binding for

(9C6) (upper) and anti-Myc (lower) to detect myc-tagged Fringe. Note that

hows that the level of the transfected Notch or Ax protein were comparable.

2 cells. The level of cell surface expression was measured by binding of rat

min. Cells were washed and AP activity measured. S2 cells and S2 cells

binding of the antibodies. The data are averages of four replicates after

otch is shown below. The two samples were run on the same gel, blotted and

removed).

Fig. 4. Comparison of Delta-induced activation of Notch signaling. S2 cells

were cotransfected with a luciferase reporter construct containing Su(H)

binding sites to monitor Notch signaling activity and a control Renilla

luciferase construct expressed constitutively by the copia-element promo-

ter. The cells were cotransfected to express Fringe and Notch or Ax[M1] or

empty vector as a control. After induction, cells were cocultured for 2 days

with Delta-expressing cells, which serve as the source of ligand or with S2

cells. The data are averages of three replicates (GSE), normalized to renilla

luciferase to adjust for differences in transfection efficiency and then

normalized to the level of Delta-induced Notch activity.

Fig. 5. Cis-downregulation of Notch and Ax[M1]. S2 cells were

cotransfected to express Notch or Ax[M1] and Fringe and to express

Serrate or Delta or with an empty control vector. The level of Notch or

Ax[M1] on the cell surface was measured by anti-HA binding. The data are

averages of three replicates after background subtractionGSD. The effects

of Delta and Serrate are normalized to the level of Notch or Ax[M1] in

control cells.

L. Perez et al. / Mechanisms of Development 122 (2005) 479–486 483

background from cells transfected with the control (empty)

vector. Interestingly, Notch expression caused a significant

level of reporter gene activity in the absence of ligand (co-

culture with S2 cells). The level of ligand-induced Notch

activity was 4-fold above the level of ligand-independent

background. The basis for the ligand-independent acti-

vations is not known but it was observed reproducibly. The

Ax[M1] protein ligand-induced activity of about 1/4th that

of wild type Notch. The magnitude of this difference is

comparable to the difference in the level of cell surface

expression of Ax[M1] and Notch (Fig. 3).

The experiments described so far suggest that the ectopic

activity of Ax[M1] in the wing disc cannot be attributed to

(1) an improved capacity of Ax[M1] to bind Delta or Serrate

or (2) to an increased relative level of expression of Ax[M1]

at the cell surface or (3) to an increased intrinsic capacity of

Ax[M1] to be activated upon ligand binding. Ax[M1] did

not behave as though it had more activity than wild-type

Notch in any of these assays.

Previous studies have shown that Notch activity can be

downregulated cell-autonomously by high-level expression

of its ligands in the wing disc (de Celis and Bray, 1997;

Micchelli et al., 1997; Sakamoto et al., 2002). It has been

proposed that a difference in sensitivity to cis-down-

regulation by ligand might explain the elevated activity of

Ax alleles (de Celis and Bray, 2000). To test this we

cotransfected cells to express HA-tagged Notch or Ax[M1]

and Fringe together with Delta or Serrate (or with empty

vector as a control). The level of receptor on the cell surface

was then determined by anti-HA binding to the transfected

cells. Coexpression with Delta reduced the level of Notch to

w17% of the level in S2 cells not expressing Delta (Fig. 5).

Coexpression with Delta reduced the level of cell surface

Ax[M1] to about 29%, perhaps indicating a reduced

sensitivity to downregulation by Delta. Serrate is thought

to be a more potent cis-antagonist of Notch than Delta

in vivo (de Celis and Bray, 2000), but we see no difference

in the effects of Serrate on Notch or Ax[M1] in this assay.

Coexpression with Serrate downregulated both Notch and

Ax[M1] to w60% of control levels. Thus, it does not appear

that the ectopic activity of Ax[M1] is likely to be a

consequence of insensitivity to cis-downregulation by

Serrate, though the possibility remains for an effect of Delta.

3. Discussion

Here, we report a series of experiments, using different

assay modes, designed to ask if the in vivo gain of function

properties of the Ax[M1] mutant form of Notch can be

attributed to the properties of the Ax[M1] mutant protein as

a receptor. When assayed as a soluble protein, the

extracellular domain of Ax[M1] bound Delta less well

than the comparable domain of Notch. When expressed in

S2 cells, full length Ax[M1] showed lower activity than

Notch in binding to a soluble form of Delta. Much of this

difference can be attributed to reduced cell surface

expression of the Ax[M1] protein. When the reduced level

of cell surface Ax[M1] expression is taken into account,

Ax[M1] showed comparable activity to Notch in terms of

ligand-induced activation of a reporter gene (using cell-

surface expressed ligand to more normally reflect the in vivo

situation). None of these experiments would suggest that

Ax[M1] has more activity than Notch in vivo. For example,

reduced cell surface expression of Ax[M1] would be more

compatible with reduced activity in vivo, rather than higher

than normal activity. These findings are consistent with

genetic analyses, which reveal that Ax alleles show some

characteristics of reduced function (hypomorphic) alleles,

Fig. 6. Ectopic expression of Serrate Delta or Fringe. (A, C, E) wild-type

wing discs. (B, D, F) Ax[M1] wing discs. (A, B) ectopic expression of

Serrate driven by ptc-Gal4. (C, D) ectopic expression of Delta driven by sal-

Gal4. (E, F) ectopic expression of Fringe driven by sal-Gal4. Wingless

protein (green). Serrate, Delta or Myc-tagged Fringe (red).

L. Perez et al. / Mechanisms of Development 122 (2005) 479–486484

even though they also have phenotypes consistent with

ectopic activation of Notch (loss of sensory bristles,

truncation of wing veins, overgrowth) (de Celis and

Garcia-Bellido, 1994; Brennan et al., 1997). For example,

Ax[M1] results in lethal neurogenic embryonic phenotypes

when combined with a deletion of the Notch locus and the

bristle loss of Ax[M1] is suppressed by increasing the

dosage of wild-type Notch. The loss of function character of

Ax[M1] is temperature sensitive, being stronger at 29 8C,

consistent with the idea that the C999Y mutation results in

an alteration in proteins structure that compromises its

function. Our biochemical assays suggest that the reduced

activity of Ax[M1] seen in some genetic tests in vivo is

likely to be due to less receptor present on the surface.

Despite the hypomorphic component to Ax[M1], this

mutant causes many effects indicative of elevated Notch

activity, including ectopic expression of Notch targets Cut

and Wg in dorsal and ventral cells of the wing disc (de Celis

and Bray, 2000; Ju et al., 2000). Many of the phenotypes are

enhanced by an increase in the dosage of Delta (de Celis and

Garcia-Bellido, 1994), suggesting that Ax could have

altered sensitivity to its ligands in vivo. As our binding

assays showed reduced binding of Ax[M1] to the ligands, it

is unlikely that the mutant receptors are able to respond to

lower concentrations of Delta or Serrate. An alternative

explanation is that Ax[M1] has lost the cis-inactivation

caused when ligands are present in the same cells as the

receptor (de Celis and Bray, 2000). In wild type discs

ectopic expression of the ligands primarily results in

activation outside the domain of expression, an effect

explained by cis-inactivation within the ligand-expressing

cells. In Ax[M1] the cis-inactivation is less-evident and

there is more widespread activation by the ectopic ligands,

although they retain their normal dorsal/ventral distinctions

with Serrate activating mainly in the ventral compartment

and Delta mainly dorsally (see Fig. 6A–D). Both Ax[M1]

and Notch show a similar degree of reduced surface

availability when they are co-expressed with the Serrate.

Ax[M1] is also sensitive to co-expressed Delta, although

less so than wild-type Notch. Given that Serrate is thought

to be a more potent cis-antagonist than Delta in vivo (de

Celis and Bray, 2000), the lack of a difference of the effects

of Serrate on Notch and Ax[M1] make it difficult to ascribe

ectopic activity of Ax[M1] in vivo to a decreased

susceptibility to cis-interaction.

The glycosyltransferase Fringe modifies O-linked fucose

residues that are attached to many of the EGF repeats within

the Notch extracellular domain, including those affected by

Ax mutations. As there are striking differences in the effects

of ectopically expressing Fringe in wild-type and Ax mutant

discs, it has been suggested that the Ax mutations could result

in altered glycosylation by Fringe. Fringe is normally present

only in dorsal cells where its modification of Notch makes it

insensitive to activation by Serrate and more sensitive to

activation by Delta (Panin et al., 1997; Fleming et al., 1997;

Johnston et al., 1997). When expressed ectopically Fringe is

able to modify Notch in ventral cells, increasing their

sensitivity to ventrally expressed Delta and preventing

activation by Serrate (Fig. 6E). In Ax[M1] discs, ectopic

expression of Fringe causes very strong ectopic activation of

Notch in the dorsal compartment with a more modest effect in

the ventral compartment (Fig. 6F). This observation is

striking and suggests the possibility of a difference in the

effects of Fringe on Ax[M1] and Notch. However, we have

not been able to measure a difference in the properties of the

Fringe-modified receptors in our in vitro ligand-binding

assays. Fringe affects Ax[M1] similarly to Notch in terms of

altering their binding to Delta. Thus, even though previous

studies have reported that the Ax[M1] and Ax[59] mutations

can interfere with the interaction between Notch and Fringe

(Ju et al., 2000), we can detect positive effects of Fringe on

Ax[M1] function. In addition, direct assays of Fringe

mediated glycosylation of two Ax mutant molecules

revealed a loss of glycosylation in only one of the mutants

arguing against this being a primary cause of the hyper-

activation of Ax proteins (Shao et al., 2003).

L. Perez et al. / Mechanisms of Development 122 (2005) 479–486 485

How can we explain the gain of function characteristics

of Ax[M1]? The fact that ectopic Fringe has such different

effects in Ax[M1] and that Ax[M1] partially rescues the

phenotype of the fng[D4] allele (de Celis and Bray, 2000)

argues that the amino-acid substitution in Ax[M1] activity

should change an aspect of Notch function that is influenced

by Fringe. One possibility is that Ax[M1] modified by

Fringe might be less sensitive to an inhibitor of Notch

activation, hence the gain of sensitivity to Serrate and Delta.

Alternatively, Ax[M1] might be very sensitive to an

activating ligand other than Serrate or Delta. A third

possibility is that there may be some component present in

the imaginal disc cells that is lacking from S2 cells and that

is required for Fringe-modified Ax[M1] to acquire its

apparent gain-of-function activity in vivo. We favor a model

in which the effects of Ax[M1] depend on some component

expressed in the wing disc, because we can find no evidence

that any aspect of Ax[M1] function as a receptor for the

known ligands is increased in a manner that could explain

the properties of this allele in vivo.

4. Experimental procedures

4.1. Plasmids and constructs

Ax[M1] was constructed by PCR amplification of a

fragment of Notch introducing the Cysteine 999 to Tyrosine

change in EGF module 25 (sequence at codon 999 TGC

changed to TAC). Notch-HA and Ax[M1]-HA were made

by inserting oligonucleotides encoding an HA epitope

(YPYDVPDYA) into the unique KpnI site between the

second and third EGF modules. Notch-AP and Delta-AP

alkaline phosphatase fusion proteins, Delta, Serrate Fringe

and Fringe NNN mutant constructs were described in

Bruckner et al. (2000). Ax[M1]-AP was prepared by

replacing the NheI-BglII fragment of Notch-AP with the

corresponding fragment of Ax[M1] produced by PCR.

The Notch reporter construct was prepared by placing the

transcription-factor binding-site and promoter regions from

GbeCSu(H)m8 (Furriols and Bray, 2001) upstream of

luciferase in pGL3 (Promega). Details of constructs are

available on request.

4.2. Binding and luciferase assays

Binding assays with AP fusion proteins were performed

as described in Bruckner et al. (2000). All assays were

performed in at least three separate experiments with

comparable results. In each case results of one experiment

are shown. The amount of DNA used for transfection was

adjusted to an equal level by addition of the appropriate

empty vector. To measure cell surface expression of HA-

tagged Notch and Ax[M1], transfected cells were induced

for 2 days, washed with HBSS containing 0.1% NaN3 (to

block metabolism and receptor recycling) and incubated

for 90 min at room temperature with rat anti-HA (Roche) at

1/500 dilution in PBS containing 0.1% NaN3, washed and

incubated for 90 min at room temperature with AP

conjugated goat anti rat IgG (Jackson Immunoresearch) at

1/1000 dilution in PBS containing 0.1% NaN3. For the

Notch transactivation assay cells were transfected with the

firefly luciferase containing Su(H) binding sites and with a

Copia-Renilla luciferase plasmid (kindly provided by Jussi

Taipale). The Promega Dual luciferase assay system was

used and levels of firefly luciferase activity were normalized

to Renilla luciferase to correct for variation in transfection

efficiency.

4.3. Flies and antibodies

AxM1 and the Gal4 drivers ptc-gal4 and sal-gal4 are

described in Flybase. UAS-Delta, UAS-Ser and UAS-Fng-

Myc are described in Diaz-Benjumea and Cohen (1995),

Bruckner et al. (2000) and Doherty et al. (1996),

respectively. Antibodies against Serrate, Delta and Wg are

described in Weihe et al. (2001), Doherty et al. (1996) and

Brook and Cohen (1996), respectively. Other antibodies are

commercially available.

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