407

Complexity of EGF receptor signalling revealed in Drosophila Matthew Freeman

The multiple roles of the Drosophila epidermal growth factor

receptor (EGFR) require that its activation is regulated

precisely. Recent work has highlighted two important control

mechanisms: the existence of multiple ligands with distinct

properties and the interaction between EGFR pathway and

other signalling pathways. The integration of signalling

pathways into networks is beginning to be understood.

Addresses MRC Laboratory of Molecular Biology, Hills Road, Cambridge

CB2 2QH, UK; e-mall: mfl @mrc-lmb.cam.ac.uk

Current Opinion in Genetics & Development 1998, 8:407-41 1

c’ Current Biology Publications ISSN 0959-437X

Abbreviations

DPP Decapentapleglc

EGFR epidermal growth factor receptor

TGF-a transforming growth factor-a

Introduction ‘l‘hc /~mc~~/~/li/0 epiderni;tl gro\vth fLlcror receptor (l<(;FK)

illlistrates ;I centrlil issiic in dc~~elopmeiital biolog); It is

used many times throughout dc\xlopmcnt but each time

it5 xti\xtion c;luscs cells to adopt different t’atcs. How can

one receptor trigger so many distinct outcomes? Ir is not

achie\~ed 1)~ acti\xring differcnc intracellular transduction

pathnxys; in e\wy cast stdicd. the principal effcctor of

R(;FR signalling is the Km/Kaf/\lAP kinasc pathu2)

[ l’.Z..i]. Hew, I dcscribc some rtxen~ advances that

explain hmv lC(;l;K can do so much.

‘Ike Imad mitline of the answer to this question is sug-

gcsted by the analysis of I<(;FIi function in the dtxcloping

/~mw~~i/~~ eye. Flcrc. K(;I:K is the main trigger of diffcr-

cntiarion of all ccl1 t)pcs (ncurond and non-neuronal).

achic\wl I))- reiteracivc acti\xtion of the receptor through-

out eye dc\~elopnien~ [A]. Since the sanic signal specifies

multiple factors, the main reason for different ou~comcs of

E(;Fli signalling in the eye is &It cells change their ‘stax’

throughout dc~~clopmenr: specificit! is not imparted by the

signal itself, rather, the point ;iI which they rccei\ c rhc sig-

nal deterniinrs their fate [S’]. ‘Ike cell stacc can be trans-

LiKxi 3s meaning that the target genes which bill be

csprcssrd in response to K(;l:K signalling. In the fly eve,

there are indrcd ;I nirniher of cell-rype-specific tr:inscrip-

tion factors (see for exlniple [h-X]) and it is the coordina-

tion of these fxtors that needs to he understood.

Ncverthcless, the fact that rhc exact specification of cell

FJtc is controlled in the nucleus does not esonerarc RGI;K

signalling from ;I role in the correct determination of cells.

I:or ii single receptor to ha\ e so many de\~eloprncntal

conwlucnces, its spatial and tcniporal regulation needs to

be precise. It is the combination of precise signalling and

;I network of interacting target gene that is crucial. I;or the

rest of this article. I descrilx \vork that Ixgins to re\xal

how the necesxlry precision of E(;I;K activation is

achir\ui. ‘I’hc receptor is notd>le for the number of dif-

fcrenr kinds of regulation to which it is subjected. Here. I

fouls only on qwcts that II~\YA de\~cloped recently -

there king sc\wal niorc coniprchcnsi\~e rc\iew on the

sul)jcct [2..3.9].

Feedback inhibition by Argos :\rgos is ;I sccrcrcd inhibitor of E:GFK [lO]. As with

K(;lTK-acri\xting ligands. the cor(: domain of Argos is an

R(;t; motif. :ilthough its inhibitory niechanisni is still

ilnresol~xxl [ 111. Iniport;lntl~, im expression is dcpenden~

on K(;I;K signalling [ 121. thcrelq forming a ncgati\xz feed-

hack lool~ - tcrnied remote inhibition [-I] - chat limits

signalling (I:igiire 1). Argos has ;I cenrral role in rrgulating

rcccptor acti\3tion. I;or exaniplc. in the kcntral cctodcrm

of the embryo. it is needed to maintain a graded activation

of the receptor [13]. In the eye, Argos lxuxnc cells from

diffcrenriating precociously [13-l 51. In rhesc t\\‘o exani-

plcs, Argos has ;I distinct role. In the wntral ectoderm it

maintains ;I gradient of E(;l.3K acciva&n \+.hcreas in the

eye ic corn~erts a pxicd signal (rhe acti~xting ligand dif-

fusing from 21 source) into ;I binary one: cells are tither acti-

\xted or nor. ‘I’his feedbacl~ inhibition is rhcreforc a

\xrsatilc mcchanisni that controls patterned acci\xtion of

the K(;FK [i’]. Intriguingly ;I similar inhibitor has IIOU’

been discoxrcci for anothcl- Z)mvp/Iilu receptor tyrosine

kinasc. the fil,roblast grm\ Ih factor reccpor (t:(;l:K) [lb’].

‘l‘hc expression of rhis factor. Sprougz is dependent on

lT(;121i signalling, csuctl~ 2s Argos is delxndent on EGFII

signalling anti thcrcfk also forms ;i ncgaC\-e fcctiback

loop. ‘l’he striking similarity in die control mechanisms of

these two receptors suggest that feedback inhibition

nicchanisnis ma) Ix \vidcsprcatl.

Multiple activating ligands of the EGFR I,ikc its maninialian coiincerpir~s. I)rm0p/IiIu EC; 1: K is

acti\xted b). niultiplc ligands (ITigure 1 ). (;urken and Spitz

arc each similar to transforming gro\\th facror alpha

(‘I’(;k-Cx) [l 7,l H]. (;iirkcn appears to bc confined to rhe

oocytr and Spiu is the principal acti~xting ligand in the

dr\.eloping fl>-. A third ligand, \‘cin, is related to the man-

malian nciirejiiilin family of K(;I:K ligands [19]. ‘I’hcse

three lipids ma)- not be the L\ hole story as there arc some

RGFK functions not accounted for and there are indeed

se\wd orher ligands in maninuls [30-2-l]. Arc the diffcr-

cnl ligands siniply inccrchangeahle v.a).s of acti\.atin,g the

receptor. or do they ha\.e different functions? In

/lrm@ilu, there is c\.idence that they arc distinct, both in

their regulation and their function. For esalnple, although

<;urkcn and Spiu arc similar and arc both anchored to the

408 Pattern formation and developmental mechanisms

Figure 1

Other ligands? e.g. Amphiregulin, betacellulin, Cripto, HB-EGF

Multiple EGFR ligands. Gurken and Spitz are both TGF-a-like, but act

in different tissues and are regulated differently-for example, Spitz

requires proteolytic cleavage from the membrane, whereas this may

not be the case for Gurken. Vein is similar to neuregulin and is a

secreted protein. It is not known whether other classes of mammalian

EGFR ligands have Drosophila homologues. Argos is a secreted

Inhibitor of EGFR signalling that forms an inhibitory feedback loop.

cell surface by a transmcmbrane domain. Spitz needs to bc

cleaved to 3 soluble fragment to he functional (rcviewcd in

[A]), probably under the control of the membrane proteins

Star and Khomhoid [ZS-2X]. ‘l’herc is no aidencc that

(;urkcn needs such cleavage - although it is not ruled

out - but it does reclkiirc the post-tr3nscriptioii~ll action of

scwral gents not needed 1)~ SpitL (for example, set [%I).

1)istinctions can also bc drau.n between Spitz and kin.

Spitz is expressed broadly throughout de\4opmcnt [ 171

and. as mcntioncd abo\,e, needs proteolytic processing.

(:onvcrsely, Lcin is expressed in a dynamic pattern that

suggests direct transcriptional control of its action and is a

secrctcd protein which may not require l”)st-translational

modification [IV]. ‘I’here arc also functional distinctions

hct\vecn Spitz and \‘ein. In the de\.eloping midgut, \‘cin

from the mesoderm induces the differentiation of underI?;-

ing endodermal cells (which express the KGFK) [W]: con-

sistent bvith this. mcsodcrmal o\wcxprcssion of \cin caIIses

endodermal defects. In contrast, mesodermal 01 errxpres-

sion of the active form of Spitz has no effect. ‘I’he reason for

these different properties is unknown but Spitz may be

unable to cross the basal membrane that separates the germ

layers whereas LTein clearly can. \‘cin is also the ligand that

acts in a different example of signalling between germlay-

ers: from myotubes in the mcsoderm to epidermal muscle

attachment cells in the ectoderm [31’]. Another tissue-spe-

cific function is found in the eye: Spitz overexpression has

dramatic consequences [4], whereas kin has no effect

(Jr1 Wasserman, iL1 Freeman. unpublished data).

In summary, the distinctions between the \ arious l<(;12K-

activating ligdnds are still not very clear but the e\idencc

is mounting that they do not ha\e identical properties.

‘I’his is true of both their regulation and their functional

properties in different tissues. ‘I’hese ohserwtions suggest

that the choice of ligand and the possible interplay

between ligands may be ;1 significant clement in control-

ling the activation of the kX;l:K.

Interaction between EGFR signalling and other pathways The analysis of K(;l’Ii function in the developing cyc of

Zhso~/lih indicated that it ~vas the key trigger of diffcren-

tiation of all cell types [-I]. There is, ho\vevcr, clear wi-

dence of interaction hetwecn EGFK signalling and other

pathwayi in several contexts (Figure &I), our understand-

ing of which is sure to grow owr the next few lears.

Pathways tend no\v to bc studied 3s discrete entities hut

they are integrated within ;I dewloping organism: sig-

nailing actually occurs in networks rather than pathu3y.

(:ertsin questions can bc asked: \\%ich other pathway

interact with E:GFK signalling? \Vhat are the devclopmcn-

tal consequences of these interactions? \\:hat are the mech-

anisms of this interplay between signaIling path\\ ays (e.g.

at what level do they intersect)?

‘lirw groups ha1.e sho\vn an antagonistic interaction

bctu,een I<(;I;K and signalling by \Vinglcss (the \Vnt-1

homologuc in D/n.rop~ih) in the larval cpidcrmis [.3?,.33*].

‘l‘hc kentral side of the laru is co\.ercd hy stripes of dcnti-

clcs, betlveen Lvhich is naked cuticle. \Vingless is required

for making the naked cuticle [.3-l]. u.hercas most of the

denticles arc dependent on E(Gl;K signalling [.32’]. ,111 cp-

dcrmal cell’s fate is influenced I>?; competition hct\vccn

\Yingless and E:(;FK signalling: lvhere \\:ingless is strong

and I<(;l;K weak, naked cuticle is made and where lC(;I;K

signalling prwailc, dcnticlcs arc made [.32’]. I:ocusing on

just one of the denticle ro\vs. O’Krefc PI N/. [.3.<‘] rcachctl ;I

similar conclusion. ‘I’he mechanism of competition

bctuazn the two pathways is not yet known.

‘1%~ Notch receptor pathwa? also interacts \f.ith l<(;l:K

signalling - as \I ith LYingless, antagonisinx its sffccts (see

re\.icw b)- 11’einmaster, this issue. pp 4.V412). l:or cun-

pie, hvhen activated Notch is expressed in the dc\,cloping

eye, it I~locks cells front bein,q triggered to diffcrcntiatc I))

Complexity of EGF receptor signalling revealed in Drosophila Freeman 409

Figure 2

(a)

1 e Wg (Larval epidermis)

Dpp _@ I--- Notch Eye, wing)

(Midgut, follicle cells) k

+ Dpp (Trachea)

63 EGFR Dpp

no?, v 1 Mad-binding site

1 CRE 1

Ubx target gene

Current Optmon tn Geneks 8 Development

lnteractlons between slgnalling pathways. (a) Interactions between the

EGFR pathway and others which are dIscussed In the text. Note that

the mechanism of Interaction and where In the pathway it occurs is not

known in most cases. (b, c) In the case of Dpp interactions, possible

mechanisms have been described:: (b) TGF-0 signalling is antagonised

by MAP kinase In mammalian cells [45’]; a simplified scheme IS shown

here. (c) Dpp and EGFR slgnalling synergise in the Drosophila midgut

and their enhancer elements are found to overlap in the Ultrabifhorax

midgut enhancer, suggesting direct interactlon at the level of target

gene control [30’]. The two elements are also close to each other in

the labial enhancer. Note that there is another level of interactlon In this

system as the expression of Vein, the EGFR ligand, is dependent on

Dpp signalllng [30’]. This illustrates how intncate the relationshlp

between pathways can be.

K(;I:R [.3.5]. ~;urthcrmore, in genetic screens for mutations ‘I’he fly has :I remarkable number of mechanisms for rep-

that interact bvith either K(;FR [.%] or Notch 1.371, genes in lating activation and signdling throllgh the K(GFR and the opposite pathway ha\x been isolated and the class of these myriad controls go sonic \~a); to explaining how such

intcruction implies mutwl antagonism. ‘I’hc relationship a widespread receptor can participate in so many distinct

between the pathuxys has been explored most in the developmental decisions. As described here. in gcncral,

de\xloping wing \.ein, where thcrc is a transcriptional specificity derives not from the signal itself - it is alwx)-s

intcrdependcnce between the pathway rather than a the same-but by ensuring that it is only recci\)ed at cxact-

direct signalling intrraction [?A’]. Another possible inter- I? the right place and time. Distinctions bet\veen the vx-

action is suggested by the findin ,q that the (:anoe protein ety of activating ligands, either in their manner of

which - like its mammalian counterpart, M-6 - binds to

Rx, is necessary for Notch signalling [39,40]. ‘I’he func-

tional significance of this observation remains to be clari-

fied but it could provide a direct point of intersection

between Notch and E(GFK signalling.

A final cxaiiple of signalling interactions with the EGFR

in\ul\u the ‘ITiF-P family member, Decapentaplegic

(I)pp). In this case there are examples of both antagonistic

and synergistic interactions, illustrating the important gen-

eral point that the relationship betlveen path\vays is

dependent on context. 1)etcrmination of tracheal founder

cells is controlled by both E:C;l:R and 1)pp signdling, each

of which determines a subset of founders [41-13.4-l’]. 13)

manipulatin,g the le\xzls of I>pp and E(;l:R signalling,

\Yappner PT N/. [4-I’] showed that there is an antagonistic

relationship between the lxlthways: cwxss signalling by

the lC(;l-R causes the loss of 11plAependcnt cells and

\icc-\wsa. ‘l’he mechanism is not addressed by this work

but there is an intriguing lprallel in niamnidian cells,

\t,herc hlAP kinase - hvhich is stimulated by E:(;FR -

can phosphorylate and thus trap in the qtoplasm, Smad-1.

a key transducer of ‘I‘(;I:-p si,qnalling [AS’] (I;igurc Sb).

‘I’he opposite relationship has been found in the de~~~lop-

ing midgut. KGFR and I)pp signalling from the niesoderm

arc both required to induce expression of the /&/(I/ gene,

which specifies endodcrmal cells [W]. Neither signal

alone can s\vitch on /c/&l and there is a strong synerg)

brt\vecn the signals. In this cast, a target enhancer (from

the I ‘/~uu~~/~ou/s gcnc) that models this synergistic induc-

tion has been well characteriscd, suggesting a mechanism

for the s) nergy [W]. \2;ithin the enh:mcer seclwnces the

binding site for \Iad (hlothcrs against 1>pp), the transduc-

cr of I>pp signalling, o\x!rlaps a (:Rl< (c.UlP-rcsponsc cle-

mcnt) which. as \vcll as being needed for I)pp response, is

also required for I<(;l~K responsi~encss. ‘I’lius it \IYILII~ appear that the two signals may in this case be intrgrated at the cnhanccr lekcl (lTigurc 2~). r)13p and EGFK ~SO seem to \vork in s)ncrgy in the determination of the cells

that form the dorsd appendages of the /~~v.copM~~ egg.

Only cells that recci\x both signals express the NI.(IN~ gcnc, \\ hich is necessary for appendage differentiation

[3h]. Prcs~~mably the /1wuz’ enhancer also has target

seqr~ences for the tux) signaIling pathways, and the relative

positions of these elements may pro\idc insight into the

signailing s)-nerg);

Conclusions and perspectives

410 Pattern formation and developmental mechanisms

rcjitllarion or their cell biological ~xopcrties, xc Iqinninfi

to envqc and these clearly affect hmv and when the recep-

tor is acti\xted. ‘Ike same is lilicly to bc true in mamnials.

E(;l;K mtagonists like Argos have not yet been disco\wcd

in vertebrates IJut the functionall\~ similar F(;t;K inhibitor

Sprouty does ha\x human honiologt~es [lh’]. again suggest-

ing that the logic of controlling these types of receptors is

\vcll conserkcd. In the psc )-car it has ken noral~le that the

interaction btmvcen the lCC;FK pathway and those of other

\vcll-k~~ow~~ signdling molecules has begun to Ix clarified.

It should be cmphasiscd that, for most of the cxunplcs

dcscribcd, the cuct nature of the interaction and ukther

it is dirccr or not is not yet known. hlort of thcsc sttldics

ha\~ IKXII carried out 1,~ genetic trchniques and :I mow

I,iochcmical appro3ch is necdcd. It is, homuxr, a safe 1x3

that all manner of interactions occur, from dirccr crosstalk

Ixxuxxn signals, receptors, or mnsduccrs, to integration at

the Ic\xl of the tqet cnhanccrs, and also the rclativel)

indirect rr;~nscriptional interdepcndencc of signalling path-

ways (implying ;I seclucntial relationship rather rhan :I

simultaneous one). ‘Ike integration of path\la);s into sig-

nailing ncrworks is a theme that is sure I0 lx cxpancicd in

the near future.

References and recommended reading Papers of particular interest, published within the annual period of revlaw, have been highllghted as:

l of special Interest l * of outstanding interest

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277:l 103-I 106. Uses an antibody that recognlses only the diphospho (activated) form of MAP kinase to map the activation of EGFR throughout development. Confirms that graded activation is seen in the ventral embryomc ectoderm and expanding rings of activation In the developing ommatidla In the eye - as predlcted by earlier work. HIghlIghts the value of the antibody as a tool.

2. Schweitzer R , Shilo B-Z: A thousand and one roles for the Drosophila EGF receptor. Trends Gener 1997, 13:191-l 96.

3. Wasserman JD , Freeman M: Control of EGF receptor activation in Drosophila. Trends Cell B/o/ 1997, 7:431-436.

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5. Freeman M: Cell determination strategies in the Drosophila eye. . Development 1997, 124:261-270. Develops a new model for eye development based on the reiterative use of EGFR to trigger all cell types. Discusses remote inhIbItIon by Argos.

6.

7.

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IO

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Complexity of EGF receptor signalling revealed in Drosophila Freeman 41 1

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31. Yarnltzky T, Mln L, Volk T: The Drosophila neuregulin homolog Vein . mediates inductive interactions between myotubes and their

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Development 1997, 124:3209-3219. EGFR slgnalling IS required for the differentiation of the antenor four rows of dentlcies II- each denticle belt. EGFR and Wingless slgnalling compete to determine cells. This work ftlls In a gap In the understanding of epidermal patterning.

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establish cell fate across the Engrailed domain in the Drosophila epidermis. Development 1997, 124:4837-4845.

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43

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Notch receptor blocks cell-fate commitment in the developing determination of dtfferent subsets of tracheal founder cells. An antagonistic

Drosophila eye. Nafure 1993, 365:555-557. relationship between the pathways IS revealed.

Price JV, Savenye ED, Lum D, Breltkrutz A: Dominant enhancers of 45. Kretzschmar M, Doody J. Massague J: Opposing BMP and EGF Egfr in Drosophila melanogaster: genetic links between Notch . signalling pathways converge on the TGF-beta family mediator and Egfr signaling pathways. Genetics 1997, 147:l 139-I 153. Smadl. Nature 1997, 389:618-622.

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Activated MAP kinase can phosphorylate Smad-1, the TGF-p transducer, and thereby prevent Its translocatlon to the nucleus. This IS a clear example of an antgonistlc Interactton between two pathways occurring by direct Interaction of the transduction components.

de Celis JF, Bray S, Garcia-Bellldo A: Notch signalling regulates vein/et expression and establishes boundaries between veins and interveins in the Drosophila wing. Development 1997, 124:1919-1928.

46. Deng W-M, Bownes M: Two signalling pathways specify localised expression of the Broad-Complex in Drosophila eggshell patterning and morphogenesis. Developmenf 1997, 124:4639-4647.

Characterises the genetic relatlonship between Notch and EGFR slgnalling I” the determination of wing veins. The pathways act In a series of feedback mechamsms that explain how vein pattern is controlled.

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Kurlyama M, Harada N, Kuroda S, Yamamoto T, Nakafuku M, lwamatsu A, Yamamoto D, Prasad R, Croce C, Canaan1 E, Kalbuchi K: Identification of AF-6 and Canoe as putative targets for Ras. J Biol Chem 1996, 271:607-610.

Mtyamoto H, Nlhonmatsu I, Kondo S, Ueda R, Togashl S, Hlrata K, lkegami Y, Yamamoto D: canoe encodes a novel protein containing a GLGF/DHR motif and functions with Notch and scabrous in common developmental pathways in Drosophila. Genes Dev 1995, 9:612-625.

Affolter M, Nellen D, Nussbaumer U, Basler K: Multiple requirements for the receptor serine/threonine kinase thick veins reveal novel functions of TGFbeta homologs during Drosophila embryogenesis. Development 1994, 120:3105-3117.

Llimargas M, Casanova J: Ventral veinless, a POU domain transcription factor, regulates different transduction pathways required for tracheal branching in Drosophila. Development 1997, 124:3273-3281.

Bier E, Jan LY, Jan YN: rhomboid, a gene required for dorsoventral axis establishment and peripheral nervous system development in Drosophila melanogasfer. Genes Dev 1990, 4:190-203.