Genes Dev. 1998 Schnepp 908 13

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1998 12: 908-913Genes Dev.Bruce Schnepp, Timothy Donaldson, Gary Grumbling, et al.

an?inhibitor

EGF receptor activator intoDrosophilaEGF domain swap converts a

References

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RESEARCH COMMUNICATION

EGF domain swap convertsa Drosophi la EGF receptor

activator into an inhibitorBruce Schnepp,1,3 Timothy Donaldson,1,3

Gary Grumbling,1 Stephen Ostrowski,1

Ronen Schweitzer,2,4Ben-Zion Shilo,2

and Amanda Simcox1,5

1D epartment of Molecular G enetics , The Ohio State

University, Columbus, Ohio 43210 USA; 2Department

of M olecular G enetics, Weizmann Inst itute of Science,

Rehovet 76100, Israel

In Drosophi la thefunction of theepidermal growth fac-tor (EGF) receptor is modulated zygotically by threeEGF-like proteins: Spitz (Spi), which is a potent activa-

tor; Vein (Vn), which is a moderateactivator; andArgos(Aos), which is an inhibitor. Chimeric molecules wereconstructed in which the EGF domain of Vn wasswapped with the EGF domain from each factor. Themodified Vn proteins behaved both in vitro and in vivowith properties characteristic of the factor from whichtheEGF domain was derived. Theseresults demonstratethat the EGF domain is the key determinant that givesDER inhibitorsand activators their distinct properties.

Received January 13, 1998; revised version accepted February

10, 1998.

The D r o s o p h i l a epidermal grow th factor (EG F) receptor

(D ER ) is a mem ber of t he ErbB fa m ily of recept or t yro-sine kinases (RTKs) and, like its vertebrate coun terparts,

controls cell differentiation, survival, and proliferation

in many tissues throughout development (Perrimon and

Perkins 1997; Schw eitzer and Shilo 1997). DER function

is m odula t ed by four ca ndida t e l iga nds ea ch of w hich

possesses a predicted EG F-like doma in. G urken (G rk)

and Spitz (Spi) are TG F-like proteins (Rutledge et al.

1992; N eum an-Silberberg an d Schu pbach 1993). grk is a

ma t erna lly a ct ive gene involved in es t a blis hing egg po-

larit y (N eum an-Silberberg and Schu pbach 1993; G onza-

lez-Reyes et al. 1995), w hereas spi is zygot ica lly a ct ive

a nd funct ions in t he embryo, a dult eye, a nd w ing (R ut -

ledge et al. 1992; Freema n 1994b). Vn resembl es th e ver-

tebrate neuregulins in that both possess an Ig-C 2 domainin a ddit ion t o th e EG F-like dom ain (Schnepp et a l. 1996).

V ein (V n) funct ions zygot ica lly in t he embryo a nd t he

adul t w ing (Schnepp et al . 1996; Simco x et al . 1996; Sim-

cox 1997; Yarnitzky et a l. 1997). H ere w e show that Vn

is a modera t e a ct iva t or of D ER s igna ling in com pa ris on

w ith Spi, w hich is a potent DER activator. Argos (Aos)

has been recognized recently as an inhibitor of the DER

pathw ay and w as t he first extracellular fact or show n t o

inhibit an RTK in vivo (Schw eitzer et al. 1995a). Aos

functions in the em bryo, adult eye, an d w ing (Freeman et

al. 1992; Saw amoto et al. 1994; G olembo et al. 1996).

Cons idera ble ef fort ha s been ma de t o unders t a nd t he

structurefunction relationships of vertebrate EG F-like

mitogens to aid in the development of ErbB receptor su-

peragonists or antagonists; how ever, to date these stud-

i es h a v e n o t l ed t o t h e d es ig n o f e ff ec t iv e f ac t or s

(G roenen et al. 1994). The fly system offers a unique

opportunity to define the m olecular basis for the distinct

properties of three nat ural ligands w ith different effects

on a recept or a nd could fa cil i t a t e t he development of

vertebrate factors w ith similar relative properties.

Spi, Vn, a nd A os a re s t ruct ura lly unrela t ed except

w ith in th e EG F dom ain (Fig. 1A). The EG F dom ain con-

tains a series of six cysteines, w hich form three disulfide

bonds t o genera t e a looped s t ruct ure, a nd a number of

ot her highly cons erved res idues t ha t a re know n t o be

required for binding a nd a ct iva t ing m embers of t he ver-

tebrate ErbB receptor family (G roenen et al. 1994). The

EG F domain s of Vn and Spi are not highly related (38%

cons erved) but ha ve more s equence cons erva t ion w i t h

ea ch ot her t ha n w i t h A os (Fig. 1A ). A ddit iona lly, t he

length of the predicted B loop that forms from t he region

betw een cysteines 3 and 4 is significantly longer in Aos

than in t he act ivatin g ligands (Fig. 1A). The low level of

sequence homology and the structural differences in the

EG F domain could account for the different effects that

t h e p ro t ei n s h a v e o n D E R s ig n a li n g. To d et e rm i n e

w het her t he EG F doma in is s ufficient t o confer t hes e

distinct properties w e generated chim eric m olecules by

exchanging the EG F domain of Vn for those of Spi or Aos

(Fig. 1A). The a ctivit y of t hese chim eras w as compared

w it h t he na t ive fa ct ors in vit ro a nd in vivo.

Results and Discussion

A c t i v a t i o n o f D E R p a t h w a y b y V n i n v i t r o

U pon liga nd binding, ErbB recept ors dim erize, cros s-

phosphoryla t e on ca rboxy-t ermina l t yros ine res idues ,

a n d t r a n s m i t a s ig n a l t o t h e n u c l eu s t h r o u gh t h e R a s /

Raf /ERK pat hw ay (Egan a nd Weinb erg 1993). Thu s, D ER

tyrosine phosphorylation is th e initial indicat ion of path-

w a y a ct iva t ion, a nd ER K phosphoryla t ion is a res ult of

t he s igna l t ra ns duct ion. Secret ed Spi (s Spi, t he a ct ive

form of Spi) a nd A os ha ve been s how n t o increa s e or

decrease, respectively, the level of DER tyrosine phos-

phoryla t ion in D r o s o p h i l a S2DER tissue-culture cells

(Schw eitzer et al. 1995a,b). We applied Vn produced by

transfected S2 tissue-culture cells to S2DER cells and

s h ow e d t h a t Vn i s a D ER a c t i v at o r a n d i n d uc ed D E R

t yros ine phos phoryla t ion in a dos e-dependent fa s hion

w it h a concomit a nt r ise in ER K a ct iva t ion (Fig. 1B ).

These in vitro results provide biochemical evidence t hat

the n ew ly discovered Vn protein, w hich had been li nked

[Key W or ds: Dr osophil a; vein; spitz; ar gos;EG F receptor; EG F]3These authors contributed equally to this work.4Present address:Department ofGenetics, Harvard Medical School, Bos-ton, Massachusetts 02115 USA.5Corresponding author.E-MA IL [email protected]; FAX (614)292-4466.

908 GENES & DEVELOPMENT 12:908913 1998by Cold SpringHarbor Laboratory Press ISSN 0890-9369/98 $5.00; www.genesdev.org


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t o t he pa t hw a y genet ica lly (Schnepp et a l . 1996; Ya r-

nitz ky et al. 1997), is a D ER ligand. A direct com parison

of t he pot ency of na t ive V n a nd s Spi in vit ro ca nnot be

ma de beca us e t he prot eins ha ve not been purif ied a nd

the absolute levels of each protein in the media are thus

unknow n; how ever, w e infer that sSpi is t he m ore potent

factor because the Vn:Spi EG F chimera has stronger ac-

t i v i t y t h a n Vn a n d b e c a us e s Sp i i s

more potent than Vn in vivo (see be-

low ).

I n v i t r o a c t i v i t y o f V n c h i m e r a s

r esem b l e s t h e f a ct o r f r o m w h i c h

t h e E GF d o m a i n d e r i v e s

Th e l e v el o f a c t i v a t i on o f t h e c h i -

meric prot eins w a s monit ored. The

Vn:Vn EG F chim era, w hich serves as

a c o n t r o l f o r t h e e f f e c t o f t h e a d d i -

t i o n a l r es id u es i n t ro d uc ed d u ri n g

c on st ru c ti on o f t h e c h im e ra s, b e-

hav ed like na tiv e Vn (Fig. 1C ). In con-

trast , possession of the SpiEG F do-

m a i n c o n ve rt e d V n i n t o a s t ro n ge r

D ER a cti vat or (Vn:Spi EG F) (Fig. 1C ).

The Vn:Aos EG F chim era behaved as

a n inhibit or , ra t her t ha n a n a ct iva t or

and caused a reduction in ERK activa-t ion res ult ing from t he l iga nd-inde-

pendent activation of DER (Fig. 1D).

Thes e res ult s s how t ha t t he proper-

ties of Vn are ch anged w hen its EG F

doma in is sw apped w ith that of Spi or

A os s o t ha t t he chim era s beha ve l ike

t he fa ct ors from w hich t he EG F do-

main is derived.

Vn:Spi EGF chim eras behave

a s s t r o n g a c t i v a t o r s

i n v i v o

I n t he embryo, ect opic a ct iva t ion of

t h e D E R p a t h w a y b y s Spi u s in g t h e

G al4U AS sy ste m [Kruppel (Kr gal 4;

U A Ssspi)] c a u s es a n e xp an s io n o f

v e n t r a l c e l l f a t e s t h a t c a n b e m o n i -

tored by expression of th e ventral cell

ma rker o rt h o d en t i c l e ( ot d ) (Wie-

s cha us et a l . 1992; B ra nd a nd P erri-

mon 1993; Schw eit zer et a l . 1995b;

G abay et al. 1996)(Fig. 2D). Ectopic

e xpre ss io n o f n a t iv e Vn (K rgal 4;

U A Sv n) caused no cha nge in t he ex-

pression of ot d (Fig. 2B). The Vn:Vn

EG F c h i m e ra (Kr gal4; U A Svn :vn

EG F) caused a very m ild expansion of

ot d expression (Fig. 2C). This slight

effect could be the result of higher ex-

pression of the transgene (cf. insets in

Fig. 2, B a nd C ). I n cont ra st , ect opic

expression of th e Vn:Spi EG F chim era

(K rgal 4; U A Svn :spi EGF) caused a dram atic expansion

of ot d expression that w as similar to that seen w ith ec-

topic expression of sSpi (Fig. 2E,D ).

In the w ing, ectopic activation of the DER pathw ay is

cha ra ct erized by t he a ppea ra nce of ext ra veins (St urt e-

van t et a l. 1993). Ectopic expression of na tiv e Vn in pupal

interveins (134 8gal 4; U A Svn) produced a mild or mod-

Figure 1. (A ) Schematic of n at ive a nd ch imeric EG F-like proteins. The dom ain struc-

ture of n at ive Vn, Spi, Aos, and chim eric proteins constructed betw een Vn and the Vn,

Spi, or Aos EG F dom ains is sho w n at the top.The manipulat ion to produce the chimeras

results in the addit ion of 4 residues f lanking t he EG F domain in each chim era (show n

only in the Vn:Vn EG F cartoon). (SP) Signal peptide: (Ig) immunoglobulin-like domain;

(EG F)EG F-like dom ain; (TM) transm embran e region. The alignm ent of the EG F domain s

of Vn, Spi, and Aos is show n below . The six con served cysteines are boxed. The spacing

betw een cysteines 3 and 4 is s ignificantly longer in Aos t han any of the other proteins.

(B) Activat ion of D ER signaling by Vn in vit ro. S2 cells expressing DER w ere exposed to

increasing concentrat ions of Vn in condit ioned medium. D ER act ivat ion as m easured by

tyrosine phosphorylation (anti-p-tyr) increased relative to DER protein level (anti-DER)

as Vn concentrat ion increased. The level of ERK phosphorylat ion (anti-dp-ERK) also

increased relative to ERK protein level (anti-ERK) as Vn concentration increased. The

m a x im u m le v el t e st e d w a s a 6 0 c on c en t r a t ion of Vn -c on dit ion ed m e d iu m , a n d t h e

preceding lanes are dilut ions that differ by 0.125 increments. (C) A c t iv a t ion of D ER

signaling by sSpi, Vn, and the chimeras. S2DER-expressing cells w ere exposed to con-

trol medium and medium conditioned w ith sSpi, Vn,Vn:Vn EG F, and Vn:Spi EG F. Con-

trol cells () show that the basal level of DER tyrosine phosphorylat ion is low relat ive to

DER protein level (anti-DER). sSpi-conditioned medium results in an elevation of DER

phosphorylation (anti-p-tyr). Native Vn and the Vn:Vn EG F chimera result in a modest

elevation of DER tyrosine phosphorylation, w hereas, the Vn-Spi:EG F chimera results in

a h igh level of D ER tyrosine phosphorylat ion. Corresponding relat ive increases in ERK

activat ion (anti-dp-ERK) w ere seen w ith the factors . Similar levels of Vn an d the Vn

chimeras w ere added to the S2DER cells as determined by Western analysis w ith anti-

Vn. This concentration w as equivalent to the highest used in the doseresponse-experi-m e n t (B). (D) Vn:Aos EG F inh ibits ligand-independent ERK a ct ivat ion. S2DER cells

exhibiting a high level of ligand-independent DER activation also had high levels of ERK

activation (anti-dpERK) relative to ERK protein level (anti-ERK). Addition of Vn:Aos

EG F results in a low ering of th e level of ERK act ivat ion.

EGF receptor activators and inhibitors

GENES & DEVELOPMENT 909


4/7

erate extra-vein phenotype, w hereas ectopic expression

of sSpi (134 8gal 4; U A Ssspi) caused a strong extra-vein

phenotype (Fig. 3G ). A direct role for Vn in normal vein

development has been show n; such a role has not been

demonstrated for sSpi but is likely to be involved (Sim-

cox et al. 1996; Schw eitzer and Shilo 1997). Ectopic ex-

pression of the Vn:Vn EG F chim era (1348gal4; U AS

v n : v n E GF) gave extra-vein phenotypes similar to those

seen after ectopic expression of native Vn (Fig. 3F). In

contrast , ectopic expression of the Vn:Spi EG F chim era

(1348gal4; U A Svn :spi EGF) produced a strong extra-

vein phenoty pe like th at seen follow ing ectopic expres-

sion of sSpi (Fig. 3H).

I n t he eye, ect opic a ct iva t ion of t he D ER pa t hw a y is

characterized by loss of ommatidia, over-recruitment of

cell types, and blistering (Baker and Rubin 1989; Free-

man et al. 1992; Freeman 1996). Ectopic expression of

na t ive V n pos t erior t o t he m orphogenet ic furrow in t he

eye disc (G M Rgal 4; U A Svn) ha d no effect on t he a dult

eye phenotype (Fig. 4E), w hereas, ectopic expression of

sSpi (G M Rgal 4; U A Ssspi) and t he Vn:Spi EG F chimera

(GM Rgal4; U ASvn :spi EGF) produced small disorga-

niz ed eyes w ith blist ers (Fig. 4G ,H). Surprisingly ectopic

expression of th e Vn:Vn EG F chim era (G M Rgal4; U A S

v n : v n E GF) also sh ow ed a st rong eye phenot ype (Fig. 4F).

Thes e in vivo da t a corrobora t e t he biochemica l da t at ha t Vn is a less pot ent a ct iva t or of D ER t ha n s Spi. A d-

dit iona lly t hey s how t he EG F doma in is a key fea t ure

t ha t dif ferent ia t es V n a nd s Spi beca us e Vn ca n be con-

vert ed int o a m ore pot ent D ER a ct iva t or i f i t s EG F do-

main is sw apped w ith that of Spi. The ability to differ-

entially regulate signaling depending on w hether Vn or

s Spi is ut i l ized m a y be one mecha nis m by w hich D ER

elicits specific cell responses during development .

Figure2. Ectopic expression of nat ive and chim eric factors in

the embryo. Each panel show s the expression of o t d , w hich is a

marker for ventral fa tes. (Insetsi n B , C , a n d E) Endogenous vn

expression in tw o ventrolateral domains and ectopic expression

of v n a n d t h e v n chimeras in a circumferential band spanning

t h e K rexpression dom ain [second t horacic segment (T2)fourth

abdominal segment (A4)]. These controls show the level of ec-

topic expression of the factors. (A ) Wild t ype. On e t o t w o row s

of cells on either s ide of the ventral midline express o t d . (B)

K rgal 4; U A Svn . There is no change in ot dexpression foll ow -

ing ectopic expression of nat ive Vn in the Kr domain. (C) K r

gal4; U ASvn:v n EGF. There is a very mild expansion of ot d

expression follow ing ectopic expression of the Vn:Vn EG F chi-

m era. This m ay be the result of higher ectopic expression of this

factor than nat ive Vn (cf . insetsi n Ba n d C). (D)Kr gal4; U A Ssspi. Ec t opic e x pr es sion of s Sp i c a u se s v e n t ra l c e ll f a t es t o

spread lat erally in T2A4 as seen by the expanded expression of

o t d . (E) Krgal4; U A Svn :spi EGF. Ectopic expression of the

Vn:Spi EG F chim era causes an expansion of ot d expression

similar t o t hat seen after ectopic expression of nat ive sSpi. Ar-

row heads in A E indicate segment T2.


the w ing. (A )Wild-type w ing show ing th e norma l patt ern of five

longitudinal veins and tw o crossveins. (B) 69Bgal 4; U A Svn .

Extra vein phenotype produced by overexpressing native Vn in

the w ing disc. The w ing is smaller than w ild type because vein

cells that replace intervein cells are more compact . (C) 69B

gal 4; U A Saos. (D) 69Bgal 4; U A Svn :aos EG F.Ectopic expres-

sion of nat ive Aos (C) and the Vn:Aos EG F chimera (D) causesloss of w ing veins and the w ings are smaller than w ild type due

to a reduction in the number of vein and intervein cells. Ectopic

expression of Vn:Aos EG F gives a milder v ein loss phenotype

than native Aos. The fusion betw een L1 (the margin) and L2 is

unlikely to be due to expression ofU A Svn :aos EGFas it is also

seen in flies expressing the 69Bgal 4gene a lone (arrow head in

D). (E) 1348 gal 4; U A Svn .(F) 1348gal4; U A Svn :vn EGF. In E

a n d F there is a moderate extra-vein phenotype produced by

ectopic expression of factors in pupal in tervein regions. (G)

1348 gal 4; U A Ssspi. (H) 1348 gal 4; U ASvn :spi EG F.In G an d

H there is a s trong extra-vein phenotype produced by ectopic

expression of sSpi and the Vn:Spi EG F chimera in pupal inter-

vein regions. All w ings are show n a t the sam e m agnificat ion.

Schneppet al.

910 GENES & DEVELOPMENT


5/7

The Vn:Vn EG F chim era appears to be a more potent

fa ct or t ha n na t ive V n in t he eye beca us e unlike ect opic

Vn, w hich had no effect, ectopic Vn:Vn EG F produced a

strong phenotype. This suggests t hat regions outside t he

EG F doma in ca n a f fect t he a ct ivit y of a fa ct or beca use

t he m a nipula t ion us ed t o crea t e t he chimera s cha nged 4

residues flanking the EG F domain (Fig. 1A). This obser-

va t ion a ls o underscores t he im port a nce of a s s a ying for

liga nd funct ion in a n umber of cell t y pes beca use s uch

modula t ing ef fect s m a y only lea d t o a pprecia ble dif fer-

ences in phenotype in some cell types.

V n : A o s EGF c h i m er a s b eh a v e a s i n h i b i t o r s i n v i v o

To test w hether Vn could be converted int o an inhibit or

by sw apping its EG F domain w ith that of Aos, w e com -

pared the effects of ectopic expression of Vn, Aos, and

t he V n:A os EG F chim era in la rva l w ing a n d eye dis cs .

Native Vn produced an extra-vein phenotype w hen ex-pressed ectopica lly in l arval w ing discs as expected for an

a ct iva t or of D ER s igna ling (69B gal 4; U A Svn ; Fig. 3B).

I n t he w ing, ect opic s uppres sion of t he D ER pa t hw a y

is cha ra ct erized by vein los s (Sa w a mot o et a l . 1994;

Schw eitzer et a l. 1995a) and ectopic expression of nat ive

A os (69Bgal 4; U A Saos) or t he Vn: A os EG F chimera

(69Bgal4; U A Svn :aos EGF) resulted in vein loss (Fig.

3C,D ). The vein loss phenoty pe associated w ith ectopic

expres s ion of V n:A os EG F w a s not a s s evere a s t ha t

caused by native Aos.

In the eye, reduction in act ivity of the D ER path w ay is

cha ra ct erized by los s of cell t y pes a nd fus ion of omm a -

tidia (C lifford and Schupbach 1989; Freeman 1994a).

There w as no observable effect on adult eye phenotypefollow ing ect opic expres s ion of na t ive V n in eye dis cs

(69Bgal4; U A Svn), b u t e ct o pi c e xp re ss io n o f t h e

Vn:Aos EG F chimera (69Bgal4; U A Svn :aos EGF) pro-

duced a rough eye phenotype w ith fused lenses similar

to, but not a s severe as, that produced by ectopic expres-

s io n o f n a t i v e A os (69B gal 4; U A Saos) (Fig. 4C,D ).

These results show that the EG F domain is a key deter-

mina nt res pons ible for t h e dif ference bet w een Vn a nd

Aos and tha t t he EG F sw ap is sufficient t o convert a D ER

activator into an inhibitor. The Vn:Aos EG F chimera is

apparently n ot as potent an inh ibitor as native Aos in the

eye or the w ing, suggesting that other regions of the pro-

teins (Vn a nd/or Aos) m ay play m odulating roles.

C o n c l u d i n g r e m a r k s

We ha ve s how n t ha t V n is a modera t e a ct iva t or of D ER

s igna ling a nd ca n be convert ed int o a s t rong DER a ct i-

vator by exchanging the Vn EG F domain w ith th at of Spi

or int o a n inhibit or by excha nging t he V n EG F doma in

w it h t ha t of A os . This demons t ra t es t ha t t he EG F do-

m ain is the key feature that gives each ligand its distinct

property. The result is important for understanding the

funct ion of t he novel ext ra cellula r inhibit or A os , a s i t

s uggest s t ha t t he EG F doma in is s uff icient for D ER in-

hibition w hile oth er regions of th e protein are not essen-

tial (but may play modulating roles). There is currently

no definitive evidence that Aos elicits its effect on DER

signaling by binding DER directly; how ever, our results

suggest that the EG F domain is a crit ical region for me-

dia t ing t h e ef fect .

The fly system , w hich can efficiently test th e function

of chim eras in vivo, m ay be a pow erful tool for precisely

defining the region w ithin the EG F domain that is char-

a ct eris t ic of a n inhibit or. Hum a n EG F chim era s w i t h

substitut ions or insertions of sequence from t he B loop of

t he A os EG F doma in funct ioned a s a gonist s ra t her t ha n

ant agonists (van de Poll et a l. 1997). N evertheless, th ese

results demonstrate t hat fly /vertebrate chimeras are able

to bind a nd elicit a response from a vertebrate EG F re-


the eye. All panels show scanning electron micrographs of adult

eyes. (A ) Wild-type eye show ing the highly structured array of

omm atidia , some of w hich are show n at higher magnificat ion int h e inset. (B) 69Bgal 4; U A Svn . Ectopic expression of na t ive

Vn in the eye disc produces eyes that are indistinguishable from

w ild type. (C) 69B gal 4; U A Saos. (D) 69Bgal 4; U ASvn :aos

EGF.(C ,D) A rough eye phenotype caused by ectopic expression

of nat ive Aos and t he Vn:Aos EG F chimera in the eye disc. The

eyes are smaller than w ild type and some om mat idia are fused

(see insets). (E) GM Rgal4; U ASvn . Overexpression of n at ive

Vn in cells behind the morphogenetic furrow apparently has n o

effect . The occasional brist le duplicat ions and depressions in

the lenses are also seen in eyes of flies expressing the G M Rgal4

gene alon e (not show n). (F)G M Rgal4;U A Svn . (G)G M Rgal4;

U A Ssspi .(H) G M Rgal4; U A Svn :spi EG F.(F,G,H) Ectopic ex-

pression of factors produces disorganized eyes w ith loss of om-

mat idia and blisters . (A H) Insetsare f ivefold ma gnificat ion of a

part of t he w hole eye im age.

EGF receptor activators and inhibitors

GENES & DEVELOPMENT 911


6/7

cept or a nd s ugges t t ha t inhibit ory s equences ident if ied

i n t h e f l y s y st e m c o ul d b e u s e d t o m o d i fy v e rt e b ra t e

fa ct ors t o funct ion a s a nt a gonis t s. The development of

ErbB antagonists has significant clinical implications, as

t hes e recept ors a n d t heir l iga nds funct ion in grow t h of

tumor cells (G roenen et al. 1994).

Materials and methods

Gener ation of chi m er ic genes

The c hi m e ra s w er e m a de by c o m bi ni ng a v n c D N A l a c ki ng t he EG F

m o t i f w i t h f r a gm e nt s e nc o di ng t h e EG F m o t i f o f v n , s p i , or aos. To

generate the cD NA lacking th e EG F motif, av nt ype 1 cDN A correspond-

ing to nucleotides 16793980 w as used as a templat e to am plify 5a nd 3

regions extending to 10 amin o acids before the first cysteine and from 10

amin o acids after the sixth cysteine, respectively. For the 5fragment, th e

primers 5-ATTAACC C TCAC TAAAG -3(this corresponds to the T3 re-

g i o n i n p B S , t he v e c t o r i n w hi ch t he v nc D N A i s i ns e rt e d) a nd 5-AC-

C C G G G AAAG TG AACTG G TG AG G CC TTG -3, w hich incorporates an

X m aI s i t e a t t h e 3 e nd, w e re u s ed. F o r t he 3 f r a gm e nt t he p ri m e r s

5-G AC TAG TG TTG C AATC TACG G C C AAATAC-3, w h i c h i n c or po -

r a t e s t he SpeI s i t e a t t h e 3 e nd, a nd 5-AATAC G ACTC AC TATAG -3

(T7 region in pBS) w ere used. Cl ones encoding the EG F mot if from v n,

spi, a n d a osw ere also generated by PC R am plification an d incorporated

5X m aI and 3SpeI si tes for insertion int o thev ncD NA lacking the EG F

motif. The primers to amplify the v nEG F motif from a v ncD NA w ere

5-ACC CG G G CCC ACG G ACCG G TCAG CC TCG -3 a n d 5-G ACTAG -

TAAAATATCTAC TG TCG G G C C -3. The primers used to amplify the

spiEG F domain from genomic D NA w ere 5-ACC CG G G AG G CC CAA-

TATTACATTCCCC-3a nd 5-G ACTAG TCAG G TAAG TATTG TCG A-

TCTC-3. The primers used to am plify the a osEG F domain from an aos

c D N A (ki ndl y p ro v i ded by K. S a w a m o t o , U ni v e r si t y o f To ky o , J a p an)

w ere 5-ACC C G G G G ACAG TCC G G G C TACAG ATATC-3 a n d 5-G A-

C TAG TTATC AC G C C G G ATTG CG TG TG -3. A l l r e a c t i o ns c o nt a i ne d

100 ng of template, 10 pmoles of each primer, 20 m M Tris-HC l (pH 8.4),

50 m M KC l , 5 m M M g C l2

, 2 m M dN TPs , a nd 5 u ni t s o f Ta q D N A p o l y -

merase (G IBCO BRL). C ycling condit ions w ere 1 cycle at 95C for 5 min,

follow ed by 30 cycles at 95C for 1 min, 60C for 1 min, and 72C for 1.5

min. For in vitro studies, cDNAs encoding native Vn and the Vn chime-

ras w ere cloned into t he X hoI a nd B amHI sites of pMK33 (Schnepp et al.

1996). For in vivo st udies, the genes w ere cloned into the X hoI a nd X b aI

si tes of t he P -element vector pUAST (Brand and Perrimon 1993).

In vitr o assays of DER signaling

Stable Dr osophila S2 cell lines w ere established that express and secrete

the Vn an d t he Vn chimeric proteins (Vn, Vn:Vn EG F, Vn:Spi EG F, an d

Vn:Aos EG F)(Schnepp et al . 1996). Receptor phosph orylat ion a ssays w ere

performed according to Schw eitzer et al. (1995b): Medium collected from

stably transfected S2 cells expressing native Vn, native sSpi, or the Vn

chimeric proteins w as added to a m onolayer of S2 cells expressing DER.

After a 5-min incubation, DER w as immunoprecipitated from the cells

and sam ples w ere run on duplicate SDS-polyacrylam ide gels and blotted.

One blot w as probed w i th anti-phosphotyrosine a ntibodies (kindly pro-

vided by M. Coggeshall , The Ohio State University) to show the level of

DER tyrosine phosphorylation, and one blot w as probed w i th anti-DER

a nt i bo di es t o s ho w t ha t t he a m o u nt o f p r ot e i n l o a de d i n e a c h l a ne w a s

s i m i l a r . The r el a t i v e a m o u nt s o f Vn a nd t he Vn c hi m e r a s w er e de t er -

mined using an anti-Vn antibody (Schnepp et al . 1996). For the dose

response experiment, Vn-conditioned serum-free medium w as concen-t r a t e d 60 by c e nt r i f u ga t i o n t hr o u gh a M i l l i p or e B i o m a x 10K c o l u m n.

The concentrate w as subsequently diluted over an eightfold range w i th

0.125 increments. ERK assays w ere performed according t o G abay et al .

(1997): C onditioned m edium w as added t o S2DER cells for 14 min. The

cells w ere lysed, and samples w ere run on duplicate gels and blotted. One

blot w as probed w i th an ti-diphosphorylated ERK (Sigma M 8159)to show

the level of ERK phosphorylation, and one blot w as probed w i th an ti-ERK

(Sigma M-5670) to show that the amount of protein loaded in each lane

w as similar. To show the l igand-independent inh ibition by Vn:Aos EG F,

S2DER cells w ere incubat ed w i th Vn:Aos EG F-conditioned m edium for

14 m i n. C o nt r o l s w er e i nc u ba t e d w it h m e di u m f r om u nt r a ns fe c t ed S 2

cells.

Tr ansgenic cell l ines

Multi ple transgenic l ines w ere established for each const ruct: 8 l ines of

U A Svn ;6 l ines of U A Svn :vn EGF;17 lines of U ASvn :vn spi EG F;a nd

12 l ines of U A Svn :aos EG F.Single transgenic l ines ofU A Saos(kind ly

provided by R. How es and M. Freeman, MRC , Cam bridge, UK) and U AS

sspi w ere examined.

Ectopic expr ession in the embr yo

To examin e the effect of ectopic expression of th e factors in t he embryo

t he U A S transgenes w ere expressed w ith the Kr gal 4 driver (ectopic ex-

pression in segments T2A4) and ot d expression w as monit ored. ot d

expression serves as a m arker for ventral cell fate. Embryos w ere col-

lected for 2 hr from a cross betw een K rgal4/ TM 3f l ies and fl ies w i th t he

native or chimeric EG F-like genes (U A Svn , U A Svn :vn EGF, U A Ssspi,

or U A S-vn:spi EGF). The embryos w ere aged for 5.5 hr at 25C (about

stage 11) and hybridized w i th a digoxygenin-labeled ot da nt i s e ns e RN A

probe. In si tu hybridization to young embryos (13 hr) w i th a v n probe

w a s u s ed t o s c re en f o r l i ne s i n w hi c h vn a n d t h e v n ch imeras w ere

ectopically expressed in theK rpattern a t sim ilar levels. Lines expressing

comparable levels of v n an d vn:spi EGFw ere found. The l ine w i th the

low est expression of v n : v n E G F w a s u s e d, bu t i t ha d a s l i ght l y hi g her

expression level th an vn an d vn:spi EGF.

Ectopic expr ession in the wing

To analyze the effect of ectopic expression of the factors in the w ing the

UAS transgenes w ere expressed w ith the 134 8gal 4o r 69 Bgal 4 drivers,

a nd t he v e na t i o n p a t t e r n w a s e x a m i ned (25C ). Whe n e c t o pi c a l l y e x -

pressed in pupal intervein regions (1348 gal 4) U A Sv n produced a mild(4 lines) or moderat e (4 lines, Fig. 3E)ext ra-vein phenotype;U A Svn :vn

EG Fproduced a mild (2 l in es), moderate (2 l in es, Fig. 3F), or strong (2

lines) extra-vein phenotype;U A Ssspi produced a strong extra-vein phe-

notype (1 l in e, Fig. 3G ); and U A Svn :spi EGF produced a moderate (6

lines) or strong (11 l ines, Fig. 3H) extra-vein phenotype. We used the

69Bgal 4 driver, w hich causes ectopic expression in the larval discs, to

test the effect of ectopic expression of U A Saos a n d t h e U A Svn :aos

EG F c hi m e r a i n t he w i ng be c a u s e t he s e t r a ns ge nes di d no t p ro du c e a

phenotype w hen ectopically expressed w i th the 134 8gal 4driver. When

ectopically expressed in the w ing U A Saos (1 l i ne t h a t s ho w s s t r o ng

expression, Fig. 3C) and U ASv n: ao sEG F(12 l ines that show ed similar

phenotypes, show n in Fig. 3D)caused loss of veins. M ost69B gal 4; U A S

vn a ni m a l s di e, bu t a f e w a du l t s s u r vi v e d a nd t he s e ha d a s t r ong e x t r a -

vein phenotype (Fig. 3B).

Ectopic expr ession in the eye

To analyze the effect of ectopic expression of the factors in the eye thetransgenes w ere expressed w ith the GM Rgal4(17C )o r 69B gal 4(25C )

drivers, and the adult eyes w ere examin ed. Eye defects w ere visible w i th

the l i ght m icroscope for fl ies expressing U ASvn :vn EGF (6 lines tested,

1 l ine show ed a w eak phenotype), U ASvn :spi EGF (8 lines tested), and

U A Ssspi (1 l ine tested). No defects w ere seen follow ing expression of

U A Sv nw i th GM Rgal4(2 lines tested) or 69B gal 4(8 lines t ested). We

used the 69B gal 4driver to determine the effect of ectopic expression of

U A Saos (1 line) and U A Svn :aos EG F(12 l ines) because th e lat ter did

not produce a phenotype w hen ectopically expressed w i th GM Rgal4. A

rough phenotype w as seen in al l l ines tested. For SEM analy sis of eyes by

scanning electron m icroscopy, on e representat ive l ine w as examin ed for

e a c h c o ns t r u c t . F l y he a ds w e re de hy dra t e d t hr o u gh a n e t ha no l s er i es

(25%, 50%, 75%, 2 100%), follow ed by 50% hexamethy ldisi lazane

(HMD S) in etha nol and 2 100% HMD S for 2 hr each. The heads w ere

air-dried overnight, sputter coat ed w i th gold/palladium, and analyz ed

w i th a P hil l ips XL30 scanning electron microscope at 20.0 kV.

AcknowledgmentsWe t ha nk M . C o g g es ha l l , M . F re em a n, R. H o w e s, K. S a w a m o t o , R.

Finkelstein and t he Indiana Stock C enter for reagents, and C . Beall and E.

G ott l ieb for cri t ical reading of the man uscript. This w ork w as supported

by th e Nat ional Science Foundation (grant 97-24078 to A.S.)and an O hio

State U niversity Alum ni R esearch Aw ard (to B.S.).

The publication costs of th is article w ere defrayed in part by paym ent

of page charges. This article must therefore be hereby marked adver-

tisement in a ccordance w i th 18 U SC section 1734 solely to in dicate this

fact .

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