Copyright 0 1996 by the Genetics Society of America

Analysis of Dominant Enhancers and Suppressors of Activated Notch in Drosophila

Esther M. Verheyen, Karen J. Purcell, Mark E. Fortini’ and Spyros Artavanis-Tsakonas

Howard Hughes Medical Institute and Departments of Cell Biology and Biology, Yale University, New Haven, Connecticut 06536

Manuscript received February 19, 1996 Accepted for publication August 7, 1996

ABSTRACT The Notch receptor controls cell fate decisions throughout Drosophila development. Truncated,

ligand-independent forms of this protein delay or block differentiation. We have previously shown that expression of the intracellular domain of the receptor under the control of the sevenless enhancer/ promoter induces a rough eye phenotype in the adult fly. Analysis of the resultant cellular transforma- tions suggested that this form of Notch acts as a constitutively activated receptor. To identify gene products that interact with Notch, a second-site mutagenesis screen was performed to isolate enhancers and suppressors of the eye phenotype caused by expression of these activated Notch molecules. We screened 137,000 mutagenized flies and recovered 290 dominant modifiers. Many new alleles of pre- viously identified genes were isolated, as were mutations defining novel loci that may function in the Notch signaling pathway. We discuss the data with respect to known features of Notch receptor signaling and Drosophila eye development.

_ _

T HE Notch pathway is an evolutionarily conserved mechanism that controls the ability of undifferen-

tiated cells to respond to specific developmental signals. It is proposed that the role of this signaling pathway is to inhibit the normal progression of differentiation in immature precursor cells. Both the ectopic expression of Notch and the inactivation of Notch signaling disrupt normal differentiation, causing cells to acquire im- proper cell fates (reviewed in ~TAVANIS-TSAKONAS et al. 1995).

The Drosophila Notch gene ( N ) encodes a -300-kD transmembrane protein. The extracellular ligand-bind- ing domain contains 36 epidermal growth factor (EGF)-like repeat sequences homologous to vertebrate EGF (WHARTON et al. 1985; KIDD et al. 1986) and three LNR repeats found in Notch and its homologues (YO-

CHEM et al. 1988). The intracellular region contains six cdclO/ankyrin repeats, OPA repeats, and a PEST se- quence (WHAKTON et al. 1985; KIDD et al. 1986; BREEDEN and NASMV~H 1987; ELLISEN et al. 1991).

To determine the consequences of Notch signaling, mutated forms of the receptor have been studied in a number of organisms (COFFMAN et al. 1993; FORTINI et al. 1993; LIEBER et al. 1993; REBAY et al. 1993; STRUHL et al. 1993; NYE et al. 1994; KOPAN et al. 1994; DORSKY et al. 1995). In Drosophila, expression of truncated products of Notch lacking the extracellular domain resulted in dominant gain-of-function phenotypes,

Corresponding author: Spyros Artavanis-Tsakonas, Department of Cell Biology, Yale University School of Medicine, 295 Congress Ave., New Haven, CT 06536. E-mail: spyros-artavanis@qm.yale.edu

‘Present address: Department of Genetics, University of Pennsylvania School of Medicine, Philadelphia, PA 19104.

Genetics 144: 1127-1141 (November, 1996)

thought to be caused by constitutive activation of the pathway. This has been confirmed by the observation that expression of truncated Notch molecules leads to the ectopic expression of the E($) bHLH genes. Nor- mal expression of these genes depends on Notch and is a direct consequence of pathway activation ( JENNINGS et al. 1994; BAILEY and P O S ~ ~ O N V 1995; LECOURTOIS and SCHWEISGUTH 1995; SUN and ARTAVANIS-TSAKONAS 1996). The phenotype caused by ectopic expression of truncated Notch was termed “activated” and contrasts that of loss-of-function Notch mutations. In vertebrate systems, a chromosomal translocation affecting the hu- man Notch locus (TAN-1) and a viral insertion into the murine Notcklike int-3 gene resulted in the production of mRNAs encoding truncated proteins that consist of mainly the intracellular domain, analogous to activated Notch forms in Drosophila. These aberrant proteins were found to be associated with leukemias and neo- plastic transformation (ELLISEN et al. 1991; ROBBINS et al. 1992).

These studies of truncated forms of Notch revealed an unexpected property of the intracellular domain, namely the presence of nuclear localization signals. Notch proteins lacking the transmembrane and extra- cellular domain are nuclearly localized in transgenic flies and transfected mammalian or Drosophila cells (STIFANI et al. 1992; FORTINI et al. 1993; LIEBER et al. 1993; STRUHL et al. 1993; ASTER et al. 1994; KOPAN et al. 1994). This finding has prompted the speculation that cleavage of Notch and subsequent nuclear translocation of the intracellular domain plays a role in Notch signal- ing. Yet, the functional significance of nuclear Notch remains elusive. In certain mammalian cells, nuclear antigens recognized by Notch antibodies have been

1128 E. M. Verheyen et a1

documented, but in Drosophila, where Notch expres- sion has been monitored throughout development, no nuclear staining was detected in any tissue (KIDD et al. 1989; FEHON et al. 1991; AHMED et al. 1995; ZAGOURAS

et al. 1995). Furthermore, the activated phenotypes as- sociated with these truncated forms do not seem to be dependent on nuclear translocation of Notch as both membrane-bound and nuclear forms produce the same cellular transformations in the Drosophila eye (FORTINI et al. 1993) and in mammalian tissue culture (PEAR et al. 1996).

Initially, components of the Notch signaling pathway in Drosophila were identified by genetic studies. These include the transmembrane ligands Delta (Dl) and Ser- rate (Ser) , the cytoplasmic protein deltex (dx) , and a group of nuclear proteins, namely Suppressor of Hair- less [ Su(H) ], Hairless (H) , mastermind (mam) , and the proteins encoded by the Enhancer of split gene complex [E(spl)] (VASSIN et al. 1987; KOPCZYNSKI et al. 1988; FLEMING et al. 1990; SMOLLER et al. 1990; Xu and ARTA- VANIS-TSAKONAS 1990; DELIDAKIS et al. 1991; BANG and POSAKONV 1992; FURUKAWA et al. 1992; GORMAN and GIRTON 1992; SCHWEISCUTH and POSAKOW 1992; Bus SEAU et al. 1994; FORTINI and ARTAVANIS-TSAKONAS 1994). The screens implicating some of these compo- nents in Notch signaling relied on naturally occurring mutations, some of which have pleiotropic phenotypic effects (BRAND and CAMPOS-ORTEGA 1990; XU et al. 1990; HING et al. 1994; X. SUN, H. HING and S. A.-T., unpublished data). Specialized nomenclature for Notch alleles exists since many were originally thought to be mutations in separate loci. These include the recessive viable alleles (facet, split, notchoid) , the dominant reces- sive lethal (Notch) and the dominant putative gain-of- function Conjuens and Abruptex ( A x ) alleles. Among A x alleles, there are two classes of viable alleles that are lethal in trans to one another, an interaction termed negative complementation, and one class that is reces- sive lethal. BRAND and CAMPOS-ORTEGA (1990) isolated mutations that modified the rough eye phenotype of split. They recovered members of the Notch pathway, Dl, Hand mum, in addition to mutations in genes not previously known to interact with Notch, but that play a role in eye development, glass, scabrous and roughened eye (BRAND and CAMPOS-ORTEGA 1990). Similarly, a screen for enhancers of the recessive viable wing pheno- type of the notchoid allele of Notch recovered known members of the Notch pathway, N, Dl and mam, in addi- tion to several mutations affecting wing development: vestigial, scalloped, Bag, clipped, cut, rudimentary and wing- less (HING et al. 1994; X. SUN, H. HINC and s. ARTAVANIS

TSAKONAS, unpublished results). A screen for ex- tragenic mutations that could rescue the negative com- plementation between two classes of Abruptex gain-of- function alleles provided a stringent test for isolating members of the Notch pathway; only N, Dl, mum, and dx were able to suppress the A x lethality (FOSTER 1975;

PORTIN 1975; XU et al. 1990). FORTINI and ARTAVANIS- TSAKONAS (1994) used a trans-heterozygous combina- tion of a Notch temperature-sensitive allele and the via- ble facet allele to reduce Notch signaling to threshold levels and searched for mutations that could attenuate signaling at the permissive temperature. This screen established a role for Su(H) in Notch signaling and demonstrated that a Su(H) gain-of-function allele could also rescue the A x negative complementation. While these screens identified a number of genes required for Notch signaling, additional components may be re- quired for the signal transduction cascade regulating cell fate determination.

In an effort to identify novel pathway elements as well as genes capable of modulating Notch activity, we de- signed a genetic screen to identify second-site enhancers and suppressors of activated Notch phenotypes. Rather than utilize a naturally occurring Notch mutation, we took advantage of the phenotype produced in flies trans- formed with dominant activated Notch constructs under the control of smenkss gene regulatory sequences (FOR- TIN1 et al. 1993). Two slightly different constructs were used. One construct, sev-Notch““, encodes a protein with the signal sequence, transmembrane and intracellular domain of Notch that produces a protein localized to the cell membrane, while the other construct, sev-NotchnW1, encodes only the intracellular sequences of the protein and encodes a protein that is localized to the nucleus. Cellular analysis revealed that overexpression of either sev-Notch construct produced indistinguishable pheno- types and blocked proper cell fate commitment; precur- sor cells expressing the constructs either adopt incorrect cell fates or differentiate incompletely (FORTINI et al. 1993). These cellular changes are reflected in the mor- phology of the adult eye, both constructs producing a rough eye phenotype visible at the light microscope level (FORTINI et al. 1993).

This activated phenotype is ideal for a second-site modifier screen since it is dominant and reflects the transmission of intracellular signals in the absence of an extracellular stimulus. Moreover, the dominant phe- notype is confined to the adult eye and is not associated with any deleterious effects on the overall viability or fertility of the transgenic flies. Notch function is nor- mally required throughout eye development, and it is therefore reasonable to expect that many of the compo- nents of the signaling pathway will be expressed in this tissue (CAGAN and READY 1989). We have performed a genetic screen to isolate enhancers and suppressors of the rough eye phenotype caused by the expression of either the membrane-bound or nuclear form of “acti- vated” Notch. Here we present the results of the screen, cataloguing and describing the modifiers of the rough eye phenotype caused by expression of activated Notch.

MATERIALS AND METHODS Drosophila handling and mutagenesis: Fly cultures and

crosses were performed according to standard procedures at

Modifiers of Activated Notch 1129

25". Male w"'~ flies were mutagenized using EMS and gamma irradiation. Two- to fiveday-old males were starved for 8 hr at 25" and then fed 25 mM EMS in a 10% sucrose solution overnight (LEWIS and BACHER 1968). Ten males were mated to 50 w"'~; +/CyO, sev-Notch""' or w"'~; +/CyO, seu-Notch"' virgin females. In addition, a separate set of males was exposed to X-ray irradiation for 7.5 min, at 5 mA, 150 kV and mated to ~ 1 1 1 8 ; +/CyO, seu-Notchnuc' virgin females. Each separate Go mass mating was named in alphabetical order, and the mutant progeny were named for the parental mating, preceded by an e or s, corresponding to enhancer or suppressor mutants, and given a numerical designation following the mating name.

F1 progeny were scored for enhancement or suppression of the activated Notch eye phenotype using a dissecting micro- scope. Candidate mutations were maintained in a CyO, seu- Notch"' or CyO, seu-Notch"" background and flies with the modified phenotype were selected for several generations. Due to the design of the screen, lethal mutations on the second chromosome became balanced over the CyO, seu-Notch chromosomes upon mating virgin mutant females to mutant males. Standard segregation analysis was done for those muta- tions not mapping to the second chromosome and these mu- tations were then maintained over the appropriate FM6 or TM3 balancer chromosomes for the Xand third chromosome mutations, respectively.

Complementation testing and meiotic mapping: Once mu- tations were mapped to specific chromosomes, their homozy- gous phenotypes were determined. Mutations that were ho- mozygous viable and had no visible phenotype were discarded. Mutants that were recessive lethal or semi-lethal or that were recessive viable with a visible phenotype were retained for further study. The balanced mutants were crossed inter se to determine complementation groups. Map positions were determined by performing meiotic recombination with dominantly marked second ( S Sp BL L 8 4 ) or third (R D Ubx Pr) chromosomes. After approximate map positions were obtained, mutant flies were tested for lethality against defi- ciency stocks in the relevant regions. All deficiency stocks used in this study are described in LINDSLEX and ZIMM (1992) or in FLYBASE (1994). The deficiency kits for the second and third chromosome were obtained from the Bloomington Stock Center and several other deficiencies were obtained from the Umea Stock Center.

To classify the novel mutations, several genetic tests were carried out. First, after determining map positions, the novel mutants were complementation tested against known genes based on observed visible phenotypes or genetic behavior. Alleles of known genes that were tested against the recovered mutants include: SofzH, E l P , S C ~ ' ~ ~ ~ ' , st?, and a translocation T(2:3)57F; bare 3L (allele E-3911), which disrupts the TBP gene. The mutants were then tested against members of the Notch pathway, listed below, in an effort to determine whether they displayed genetic interactions with other Notch pathway members. This would provide strong evidence for inclusion of novel genes in the pathway. The alleles of Notch athway enes used in this study were as follows: DL'", D&', SU(H!"~, S U ( H ) ~ ~ , dx', n~arn'~"~, H', E(sPL)Bx2', Sdzp4 , Ax". All of these mutant alleles are described in LINDSLEY and ZIMM (1992) except for SU(H)'~''~ (FURUKAWA et al. 1992), S U ( H ) ~ ~ (FORTINI and ARTAVANIS-TSA- KONM 1994) and (GINIGER et al. 1994). We have identi- fied the following Pelement mutants as insertion alleles that affect kismet: 1(2)07812, 1(2)s03527, 1(2)s04771, 1(2)s04793, L(2)02532 and 1(2)rL432 (Berkeley Drosophila Genome Proj- ect; our unpublished data).

Sectioning of adult retinas: Plastic sectioning of retinas was performed according to TOMLINSON and READY (1987). Im-

ages were collected using a Leitz Orthoplan 2 microscope and converted into Adobe Photoshop for image processing.

Mi~~oscopy: Eye phenotypes were scored and classified on a Wild M10 dissecting microscope. For scanning electron mi- croscopy, adult flies were dehydrated in increasing concentra- tions of ethanol (25,50,75,95,100, loo%), mounted on EM stubs and allowed to air dry. Scanning electron micrographs were obtained using an ISESS40 electron microscope.

RESULTS

Truncations of the extracellular domain of Notch result in phenotypes that mimic gain-of-function muta- tions and are therefore thought to reflect the constitu- tive activation of the Notch receptor. We have pre- viously shown that the expression of either membrane bound ( Notchat) or nuclear (Notch""') truncated forms of Notch, expressed under the smenless promoter cas- sette, results in a rough eye phenotype (henceforth re- ferred to as the activated phenotype; FORTINI et al. 1993). Although the two phenotypes are the same at a cellular level, we decided to screen separately for mod- ifiers of each. We reasoned that if cleavage of Notch and its subsequent nuclear translocation is part of the normal Notch signaling mechanism, then the genes identified as modifiers of the phenotypes associated with the expression of the membrane bound us. the nuclear form may define different functions of Notch. The severity of the activated phenotype for each con- struct differed in various transgenic lines, presumably due to position effects at the genomic insertion site. In the case of both sm-Notch"" and sm-NotchnUc1, a line displaying a rough eye phenotype of intermediate sever- ity was selected to allow the recovery of both suppressors and enhancers (Figures 1 and 2). The screen was de- signed to recover dominant second-site mutations in genes that display a dosage-sensitive interaction with the activated phenotype. It was important to be able to recover modifiers as heterozygous mutations since many of the important signaling molecules in the Notch pathway may be required throughout development and would mutate to homozygous lethality.

To perform the screen, w1118 males were mutagenized with either EMS or X-rays and mated to females carrying an activated sm-Notch construct on the Cy0 balancer (Figure 3). Activated Notch constructs inserted onto Cy0 were chosen to prevent recombination between the transgene and new mutations as well as to preclude homozygosity for the insert. Progeny carrying the Cy0 chromosome that displayed either an enhanced or sup- pressed eye phenotype were selected. All phenotypic classification of modifiers was done using a dissecting microscope, which permits the assessment of the eye shape and color as well as the reflectivity of incident light from the irregular lens surface of the eye (Figure 2). Suppressor mutations reduced the external roughness of the eye, in addition to causing the overall eye shape to become both rounder and larger, and

1130 E. M. Ver

FIGURE 1.-Adult eye phenotype of sa-Nact. Scanning elec- tron micrographs (SEM) of wild-type eye (A and C) and Cy0,- sev-NotchnUC1/+ (B and D) eye. Expression of both the sev- Notchnuc1 and sev-Notch"' truncated Notch protein result in an intermediate rough eye phenotype. Anterior at left; dorsal at top.

less refractile (Figure 2D). Partial restoration of the crystalline structure seen in the wild-type eye was also generally observed. Second-site enhancers resulted in increased roughness and varied greatly in severity. The shape of enhanced eyes was usually smaller and flatter than the activated Notch eye phenotype; the eyes also appeared more glossy or shiny (Figure 2, E and F). These criteria allowed us to group the mutants into similar phenotypic classes. The eye phenotypes were also examined using a scanning electron microscope. This method was not as effective for classifymg the mod- ifiers as was the dissecting microscope, although high magnification views of the external eye morphology did distinguish between enhancers and suppressors based on the lens morphology. To determine whether the external phenotype correlates with modification of the internal cell fate transformations seen with the activated Notch phenotype, we sectioned representative en- hancer and suppressor mutations. Expression of trun- cated Notch under the smenless promoter causes cell fate transformations that lead to four outer and two inner photoreceptors, in addition to cone cell defects (FORTINI et al. 1993; Figure 2G). A suppressor mutation, in this case a loss-of-function Su(H) allele, partially re- stores the photoreceptor organization, so that most om- matidia possess six outer and one inner photoreceptor (Figure 3H). Enhancer mutations cause numerous de- fects that disrupt the order and organization of the ommatidia (Figure 2, I and J). Thus, in general the

,heyen et al.

mm FIGURE 2.-Phenotypic classification of enhancer and sup-

pressor mutations. All novel mutants were scored for modifi- cation of the sa-Nact phenotype under the dissecting micro- scope, by evaluating the reflection from the light source and gloss of the eye surface, which reflect the overall degree of roughness. (A) Canton-S, (B) sev-NotchnUc1, (C) sm-Notchmt, (D) Su(H)"o'O/sev-Notchnucl, (E) Su(H)eBCII/sev-Notchacl, (F) Egfr"wz/ sev-NotchnUc1. In addition, plastic sections of adult retinas were examined to determine the arrangement of the photorecep- tor cells. (G) The sev-Notch""" phenotype (FORTINI and ~ T A - VANIS-TSAKONAS 1993), (H) Su(H)"O1o/sev-Notchn"cl, (I) mum814/ seu-Notchnucl, (J) + /sev-Notch""'; Dley4/+.

effects we observe externally are accompanied by simi- lar effects on the underlying cellular architecture of the adult eye.

We screened 137,000 F1 progeny (48,000 with sm- Notchact and 89,000 with sm-NotchnuL) and recovered 290 mutant lines. These were mapped using standard segre- gation analysis and balanced. Complementation testing was carried out between all mutations on a given chro- mosome. Failure to complement was scored either by lethality or by a visible phenotype in trans-heterozy- gotes. Seventeen complementation groups consisting of two or more alleles were mapped to the second chro- mosome, 11 groups to the third chromosome, and two were mapped to the Xchromosome (Tables 1 and 2) . Meiotic recombination mapping was performed to map the autosomal complementation groups to a chromo- somal interval and deficiency stocks were used to fur- ther map the mutations (Figure 4).

The criteria for further analyzing a particular mod- ifier were the identification of more than one allele and/or an interaction between the modifier and muta- tions in other elements of the Notch pathway. The com- plementation groups were subjected to various genetic

Modifiers of Activated Notch 1131

A. EMS/X RAY

i B. + + w- ;

CyO, sm-NotchnUCi fwfl ;+ x w-;+ ; + w- ;

CyO, sev-NofchaCf lw+l ;+ x w-; + ; +

F1 w'*; * * + +

CyO, sev-NotchnUC' [w+] ' + . - F1 w- *;

* * + + CyO, sm-NofchnCt [w+1 ' +

. -

FIGURE 3.-Schematic representations of the two strategies of the screen. (A) w'"' males were mutagenized with either EMS or X-rays and then mated to females carrying the sm-NotchnUd construct. F1 progeny (89,000) were screened and dominant enhancer or suppressor mutations were selected. (B) w''" males were fed EMS and then mated to females carrying the sm- Notch"' construct. In the F1 generation 48,000 progeny were screened and dominant enhancer and suppressor mutations were selected. Candidate mutations were isolated and further characterized as described in MATERIALS AND METHODS.

tests in an effort to determine whether they interacted once and those will not be discussed further here. In with known Notch pathway genes (see MATERIALS AND most cases where a particular complementation group METHODS). The results of these interaction tests are was represented by a large number of alleles, mutations discussed separately below for each mutant group. In were recovered using both sm-NotchaC1 and sm-NotchnUC'. general, only those mutants for which more than a sin- In some cases where a complementation group con- gle allele was recovered in the screens will be consid- tained only a few alleles, mutants were not recovered ered here. Numerous mutations were recovered only using both activated Notch forms. Mutations that were

TABLE 1

New alleles of known genes

M.a? No. of alleles"

Gene Abbreviation poslhon X-ray EMS Alleles

Enhancers Notch Delta

deltex mastermind SupPrssor of Hairless Star

EGF Receptor

Son of seuenless pointed scabrous kismet

longitudinals lacking glass

Notch Suppressor of Hairless mastermind TATA-binding protein string

Suppressors

3C7 92A2

6A-F 5OC20-23 35B3C1 21E1-2

57F

34El-2 94E

21B6-7

47A11-14

49C2-D4

91A1-2

3C7 35B3-Cl 5OC20-23 57F 99A

2 37

1 5 1

13

7

5 3 1

12

3

1 3 1 4 2

eAS7bb, eUlO' eAl, eA7, eAR5, eAR6, eAZ5a: eDL3, eDW2, eK12,

eM4', eQ3', ePlO', e m ' , eAI3', eAB2', eAI9', eAZl', eAU4', eAL3', eANgb, eAWb, eAZ18', eAYZ', eAVl2', eM9', eAN13', eAR3', eAU2', e m ' , eAC2', eAT2', eBEl', eBSl', eC5, eCY4, eD6, eE4, eK1, eM1, e04, eAH5: eB3, eCX2

eAN1 ' eCI6, eF14, eA6', eAl', eA0l' eBCl1 eE6, eTV, eQlO", eAS5, eCJ9, eD03 , eA03",

eAG4', eAG5', eAQ3', e m 7 4 eAA24 e07', eAN5', eALI1, eW4

eGl, eAW2', eEP3, eCH2, eEM2, eA06, eBP3', eCP7, eCS15

eJ2, eCS3, eDN4, eCN6, eN6' eAS7ab, eT13', eAL13' eCS5 eG2, eJ6, eK15, eDV6, eE4, eFA2, eCM2, eDO1,

eES2aspo"', eCF3', eDZ6, eCQl, eAK4' eP2a'

sAR5' sA21, sK2, s o l 0 sAX8' sC4, s012, sEU4', sBTl', sP5ab, sIIOb sM3, sCG4', eAS7a'

eAL7: eCV2, eP18', eI7', eB15', eBA8', eBD1O'

spont, a mutation that arose spontaneously in an existing stock.

*Indicates which alleles were recovered in the sm-2Lpc' screen, as shown in Figure 3B. 'Denotes X-ray-induced alleles.

Number of alleles isolated for each mutagen used.

1132 E. M. Verheyen et al.

TABLE 2

1

10

1

2 1

1 2

2

Novel enhancer and suppressor mutations

No. of alleles" Complementation

group Map position X-ray EMS Alleles

Enhancers warthog (&) 32F1-3; 33F1-2 2 1 eAM4, eERl, eASl adironduck (a&) Third chromosome 2 eN9, sDU2 mango (mng) 22A5-6 1 eD9' iron felix (qx) 34C; 34E3 2 eAQ3, eC03 WII 54B1-1 1 eBBY, eEMl Ix Second chromosome 2 eAl3, eJ11 aigma (egm) Second chromosome 21 eDY2, eCP6, eBS4", eABG", eDNl, sAHl", SAH~', @Ic,

sDP4, sDZ1, sY3, sEHl", sDC9, sCS8, SLY, sP2: sEL1, sI2, eAI6', eAM2", eCN3, eC08, eAO5', eCU1, eCW3, eB3, eCS3, eCS19, eCR2, eD6a, sF2a

XII Second chromosome eCP12, eCR3, eN4: eCP9 XIII Second chromosome eAV9', eAH7* xv 75B.3-6 75B10 3-eAFlc, 4-eCN5, eAN4' XW 84A45 leCN5, sAL4' xx Third chromosome eAKl2 ', eAV4' XXIII Third chromosome eBDl', eBDY, eQ7' eH3 60A7 eH3 eK6 42B4C1; 42E eK6 eZ6 52C; 53F eZ6' XXN Third chromosome eBUl', eBG4'

snaggh (sng) 3R 2 sU4, sDX1, sK1, sAK12" XN 57GD 2 su2*, SC12' m z z 63GD 2 sCI1, sCM4

'SAB1 52A12; 52D3 1 SABl' SAM16 37D38A 1 S A M I ~ ~

Suppressors

1 1

a Number of alleles isolated for each mutagen used. ' Indicates which alleles were recovered in the smNotch"' screen, as shown in Figure 3B. 'Denotes X-ray alleles.

found to be alleles of genes known to be involved in Notch pathway genes: Mutations were recovered in Notch signaling will be described first. Then, mutations many of the genes previously shown to be associated in other known genes will be discussed, followed by a with the Notch pathway. New alleles of Notch, Delta, description of novel complementation groups. For the deltex, mastermind and Suppressor of Hairless were recov- sake of clarity, both of the activated Notch constructs ered. Two classes of Notch alleles modified sm-Nuct. First, (sacNotch"" and sm-Notch"") will be jointly referred to two Notch loss-of-function alleles were found as en- as sm-Nact, unless otherwise indicated. hancers of smNuct. These mapped to the X chromo-

N d r " 0

FIGURE 4.-Mapping of enhancer and suppressor mutations. Exact or approximate cytological locations of complementation groups are shown. Enhancer mutations are shown above the line representing the chromosome, while suppressors are shown below the line. Known members of the Notch pathway are depicted in red, new alleles of other known genes not implicated in Notch signaling are shown in green. Novel complementation groups are shown in blue. Unmapped mutations are not shown (see Table 2).

Modifiers of Activated Notch 1133

some, were homo- and hemizygous lethal and displayed the dominant visible phenotypes characteristic of N/+ flies, namely wing notches and an increased density of thoracic microchaetae. Second, one lethal Notch A h $ - tex allele, termed A F 4 was isolated as a strong suppres- sor of the activated eye phenotype. ApN4/+ heterozy- gotes displayed the shortened longitudinal wing vein IV and the bristle loss phenotype associated with these gain-of-function Notch alleles (WELSHONS 1971; FOSTER 1975; PORTIN 1975). Further analysis revealed that Ax”N4 belongs to the “Notch suppressor” class of A x alleles, as defined in PORTIN (1975), since it is lethal in combination with A#’, but viable in combination with

(data not shown; LINDSLEY and ZIMM 1992). Forty-two new alleles of Delta (Dl ) were recovered as

enhancers of sev-Nart. The enhanced phenotype seen in sa-Nact; Dl/+ flies was characterized by a small eye with a glossy, flat surface, often containing necrotic black spots (data not shown). This phenotype varied little among the 42 alleles, which constitute a group of partic- ularly strong enhancer mutations. All of these mutants have the characteristic Delta wing phenotype as hetero- zygotes and are homozygous lethal. They were mapped to the third chromosome and failed to complement Dl loss-of-function alleles and one another.

Two classes of mastermind (mum) alleles were recov- ered. Five alleles enhanced the rough eye phenotype, while one suppressed it (Figure 5). The enhancers were determined to be loss-of-function alleles by several crite- ria. They are all homozygous lethal and fail to comple- ment the mum loss of function allele, mam”-”5. It has been previously shown that mum hypomorphic alleles, in a double heterozygous combination with a Suppressor of Hairless gain-of-function allele (SU(H)“~), show a highly penetrant wing nicking phenotype (FORTINI and ARTAVANIS-TSAKONAS 1994). The five enhancer alleles recovered in this screen were able to enhance the Su(H) wing phenotype (Figure 6). The one strong suppressor allele of mum is presumed to be a rare gain-of-function allele, based on the fact that it maps to the mum region of the second chromosome and fails to complement mum loss-of-function mutants. In addition, it displays the opposite genetic interaction with activated Notch as do the loss-of-function mum alleles. It shows no en- hancement of Su(H)wing phenotypes (data not shown).

Newly recovered Su(H) alleles also fell into two classes (Figure 5). We recovered three mutations that acted as very strong suppressors of the sev-Nact rough eye, and restored the ommatidial array to a nearly wild-type con- figuration. These recessive lethal alleles map to the sec- ond chromosome and fail to complement the Su(H) loss-of-function allele SU(H)’~’’~. An allele of Su(H) that enhanced the sev-Nact rough eye was also recovered. This allele, termed Su(H)”’””, exhibits all of the genetic interactions that were previously attributed to gain-of- function alleles of this locus, including the ability to cause wing nicks in a double heterozygous combination

with mum loss-of-function alleles (Figure 6; FORTINI and ARTAVANIS-TSAKONAS 1994). The wing nicking is not observed in a mam/Su(H) loss-of-function double het- erozygote (Figure 6). The ability of these new mutations to suppress the Hairless ( H ) bristle loss phenotype was quantified (Table 3). As expected from previous analy- ses, the loss-of-function Su(H) alleles suppressed the H phenotype, while the gain-of-function allele enhanced the H bristle loss (SCHWEISGUTH and POSAKONY 1992; FORTINI and ARTAVANIS-TSAKONAS 1994).

A single allele of deltex ( d x ) was recovered as an en- hancer of activated Notch. It maps to the Xchromosome, fails to complement the dx’ allele, and displays the dx recessive wing vein phenotype, held-out wing phenotype and rough eye phenotype (XU and ARTAVANIS-TSAKONAS 1990; GORMAN and GIRTON 1992; LINDSLEY and ZIMM 1992). Like all known loss-of-function dx alleles, the new allele, termed d P N ’ , is homozygous viable.

Novel alleles of previously known genes: After map- ping and phenotypic analysis of the modifier mutations, a number of the complementation groups were found to be new alleles of known genes not directly implicated in Notch signaling. Seven of these genes (E&, Sos, S, sca, gla, stg, and pnt) are known to be involved in eye development and were recovered as enhancers of acti- vated Notch.

Eight lethal mutations affecting the epidermal growth factor receptor (E&) gene were recovered as enhancers of sm-Nuct (Figures 2 and 7). All fail to complement one another and map to the second chromosome. Rare escaper trans-heterozygous flies display phenotypes char- acteristic of loss-of-function Egfrhemizygous flies, namely very rough eyes, wing vein gaps and missing ocelli (PRICE et al. 1989). The new alleles failed to complement the deficiencies Df(2R)PuDl7 and Df{2R)PKlI, in addition to an Egfr null allele, t@2Lh5 (LINDSLEY and ZIMM 1992).

Five alleles of Son of sevenless (Sos) were isolated based on their ability to enhance sev-Nact. These mutants were homozygous lethal and mapped to cytological position 3435 on 2L. Rare hemizygous escaper flies (in trans to Df(2L) ep8) displayed very small, flat, glossy eyes and wing vein gaps, phenotypes that are seen in some Sos allelic combinations (ROGGE et al. 1991; J. ROOTE, per- sonal communication). The new mutants failed to com- plement the SOS“~ allele and were able to completely suppress the dominant eye phenotype produced by the hypermorphic Ellipse allele of E& a property that has been previously attributed to loss-of-function Sos alleles (ROGGE et al. 1991; data not shown),

Sixteen Star ( S ) alleles were recovered as strong en- hancers of sev-Nuct (Figure 7). These were mapped to cytological position 21E on the second chromosome and failed to complement a lethal Star allele. Heterozy- gous Star flies displayed a mild rough eye phenotype, while homozygous Star animals were inviable (HE- BERLEIN et al. 1993; KOLODKIN et al. 1994). The effect of a Star mutation on the eye phenotype of activated

1134 E. M. Verheyen et al.

FIGURE 5.--Enhancement and suppression of the sm-Nnct phenotype by mnm and Su(H) mutations. New mnstmin.d alleles are shown in A-D: (A and C) CyO, sev-Notchmt/mam"S", a putative gain-of function allele, (B and D) CyO, sm-Notct~"""/mamr~'~, a loss- of-function allele. New Suppressor of Hairkss alleles are shown in E-H: (E and G) CyO, s,Notch"""/.~u(H~"'", a loss-of-function allele, (F and H) CyO, sar_Notchnrt/Su(H~"'"', a gain-of-function allele. Anterior at left; dorsal at top.

Notch was severe; the eye size was dramatically reduced and the overall texture was glassy, with black necrotic spots in a field of fused ommatidia.

One allele of scubrom (scu) was isolated as an en- hancer of the activated Notch eye phenotype. This allele was detected based on its homozygous phenotype, namely very rough eyes and duplicated and split macro- chaetae (MLODZIK et al. 1990). The mutation failed to complement the scu allele S C U " ~ ' ~ ' . One allele of g h s was recovered as an enhancer of the sev-Nuct eye pheno- type. This recessive viable allele was mapped to 91A1-2 and as a heterozygote strongly enhances the rough eye phenotype of smNuct.

A complementation group consisting of two alleles (sM3, sCG4) was mapped to 99A on the third chromo- some and is uncovered by Df3R)3450. These lethal al- leles suppress the eye phenotype caused by the expres- sion of activated Notch. Trans-heterozygous flies have a mild rough eye defect and are missing many mac-o- chaetae on the head, thorax, scutellum and wing mar- gin. The bristle phenotypes are reminiscent of Hairless loss-of-function or Notch Abruptex phenotypes, although sM3/Huirless trans-heterozygotes are viable and have a H/+ phenotype. The two alleles fail to complement several mutations in the cell cycle gene string (stg) and the trans-heterozygous flies display the same bristle loss phenotype as seen in sM3/sCG4 trans-heterozygotes (EDGAR and O'FARRELL 1989; TEARLE et ul. 1994). One

pointed allele, described below, carries a secondary mu- tation in string that displays the same bristle loss pheno- type as sM3 and sCG4 in combination with various stg alleles.

A group of three lethal enhancers mapped to the right arm of the third chromosome and was found to be allelic to the pointed (pn t ) mutation. These mutants have a strong enhancer effect on smNuct eyes. Two of the alleles produce rare trans-heterozygous escaper flies that have extremely tiny, abnormal eyes, a phenotype associated with pnt loss-of-function alleles (SCHOLZ et al. 1993). One of the pnt alleles was found to have a second mutation on the third chromosome in the string gene, as mentioned above. The other two pointed alleles fully complement string loss-of-function mutants.

The seven genes described above have all been shown to be required for eye development. In addition, we recovered new alleles of five other known loci. First, a lethal complementation group of six alleles was mapped to the 57F region of the second chromosome. These mutants failed to complement a chromosome carrying an inversion that affects the gene encoding TATA-binding protein (TBP; D. WASSERMAN and G. M. RUBIN, personal communication). Heterozygosity for the putative TBP mutants was able to strongly suppress the rough eye phenotype of seu-Nuct. Second, five alleles of the gene longitudinuls lucking (lolu) were isolated as enhancers of Notch. They were mapped to the interval

Modifiers of Activated Notch 1135

FIGURE &-Genetic interactions between Su(H) and mum alleles produce wing nicking. The double heterozygous com- bination of a mum loss-of-function allele and a Su(H) loss-of- function allele displays a wild-type wing (B; compare with wild type in A). A novel gain-of-function Su(H) allele, Su(HY""", in combination with a mam loss-of-function allele produces a fully penetrant wing nicking phenotype.

47A-F on the second chromosome using a deficiency for this region (kindly provided by I. RERAY and G. M. RUBIN) and identified as new lola alleles by their failure to complement lola22112 (SEEGER et al. 1993; GINICER et dl. 1994). Third, a large complementation group of strong enhancers was found to be allelic to the kismet gene. The mutation was mapped to 21A and failed to complement a P element allele of kismet ( ki8"767. Dur- ing the analysis and mapping of kismet, we identified several additional alleles of kismet in the Berkeley Dro- sophila Genome Project P lethal collection (see MATERI- ALS AND METHODS).

Novel complementation groups: In addition to re- covering enhancer and suppressor mutations in known genes, including members of the Notch pathway and

TABLE 3

Effect of novel Su(H) alleles on Hairless bristle loss phenotype

Genotype No. of macrochaetae

+/+; HI"/+ 26.45 t 2.48 SU(H)"\~' "/+; HI/+ 36.35 2 1.21 Su(Hy" "/ + ; HI/ + 36.00 2 2.28 Su(HY'""'/ + ; HI/ + 20.8 t 2.5 +/+; +/+ 40

The effects of different Su(H) alleles on the dominant Hair- kss bristle phenotype are shown, indicating bristle site occu- pancy for the 40 major dorsal macrochaetae of the head and thorax in flies having the second chromosome heterozygous Su(H)genotypes listed in column one in combination with the third chromosome heterozygous genotype HI/+. For each genotype the mean 2 SEM were calculated from bristle counts on 40 male flies.

" A dominant loss-of-function H allele.

'A gain-of-function Su(H) allele. Two separate loss-of-function alleles of Su(H).

other loci thought to function in eye development, we recovered many modifier mutations that define new, previously uncharacterized loci. These groups consist of lethal or viable mutations with visible phenotypes. The new groups and available mapping information are presented in Table 2 and Figure 4.

A large group of 31 modifier alleles was recovered only as modifiers of the smNotchn"C1 construct, and was named enigma ( e p ) . The lethality of several indepen- dent alleles was mapped to the base of the right arm of chromosome ZZ, but was not uncovered by any of the deficiencies tested. The eye phenotype of this group is difficult to classify: the overall shape and pattern of the eye is larger and fuller, suggesting that the mutation suppresses the effect of activated Notch. However the eye appears more glossy than seen in sm-Notchn"C', which is a general property of enhancer mutants. The recovery of this large class of modifiers from only the nuclear form of activated Notch raises the possibility that the gene represented by these alleles may reflect a unique property of the nuclear form of Notch.

Two lethal mutations that strongly enhance the acti- vated Notch rough eye phenotype were named iron felix ( i f x ) (Table 2). Trans-heterozygous flies have reduced viability and escapers have very distinct phenotypic abnor- malities. These include shriveled and smaller wings that are held out from the body, abnormal wing veins, missing macrochaetae on the wing margin and mild notching on the wing margin. In addition, the eyes are very rough and small. The alleles are semi-lethal in combination with Df(2L)etyx, but viable with Df(ZL)b84h50 and Df(ZL)eP"f', placing the locus in the 34C;34D3 interval.

The complementation group named snag@ (sng) maps to the third chromosome and contains four alleles that are suppressors of sm-Nact (Figure 8; Table 2). The four alleles are homozygous lethal, and heterozygous

1136 E. M. Verheyen PI 01.

flies from three of the alleles display varying degrees of wing notching in a T M 3 background. This suggests that heteroygosity for sngmay be enhancing a cryptic S m a t p allele on the T M 3 chromosome (R. FLEMING, personal communication). Complementation testing with Dplk revealed that the trans-heteroygotes of sn,g alleles and the Dl allele, Dl'", were lethal. In contrast, sng mutants were viable and phenotypically Dl/+ in trans to Df(3R)Dl-X43, a deficiency that removes the I l l locus, suggesting that the sng mutants are not novel alleles of Dl, but that sng displays an allele-specific interaction with I l l 5/'.

The complementation group on the third chromo- some named adirondaclz (a&) consists of two alleles, one enhancer (Figure 8) and one suppressor (Table 2). Both alleles are semi-lethal, and rare homozygous flies display rough, small eyes and several wing pheno- types. The wings are rounder and shorter than wild type and are held out at an angle from the body. Ectopic cross vein material is seen between longitudinal veins IV and V, which appears to be forming an extra short longitudinal vein. In addition, a second cross vein is usually seen between the ectopic vein and LV.

The complementation group named zunrthog ( 7 m - t )

I

FIGURE 8.-Modification of sw- Notch""" bv novel second-site en- hancer and suppressor mutations. Scanning electron micrographs of adult eyes with the following genc- types: (A and D) QO, sn&"~ch"""/ +, (I3 and E) +/CyO, smNo/ch"""; P N ~ / + (an enhancer mutation from the adirondack complementation group), and (C and F ) +/CyO, sm- IVO/CIL'"'~'; sDXI/TM3 (a suppressor mutation from the snnRRIp group). Anterior at left; dorsal at top.

Modifiers of Activated Notch 1137

has a strong enhancing effect on sev-Nact and was mapped to the region 33B2-3;34A1-2 on the left arm of chromosome Il, by its inclusion in Df(2L)prdl. 7 (Ta- ble 2). Two alleles of this group are homozygous lethal and one is semi-lethal. The homozygote escapers and heteroallelic progeny have very short abnormal macro- chaetae.

Table 2 summarizes the remaining mutants recov- ered in these screens. These include groups that consist of homozygous lethal alleles, that have not yet been mapped or further characterized as well as six single hits that have been cytologically mapped. The re- maining single hit lethal mutations (-60) are not allelic to each other, have not been characterized further and are therefore not listed in this paper.

DISCUSSION

The Notch receptor mediates the specification of nu- merous cell fates during development in Drosophila. Studies of expression patterns, mutant phenotypes, and developmental consequences of unregulated receptor activation have implicated this protein in a general mechanism of local cell signaling that includes interac- tions between equivalent cells and between different cell types (reviewed in ARTAVANIS-TSAKONAS et al. 1995). Genetic approaches have been powerful tools in the analysis of the Notch pathway and have identified or strengthened the previous identification of several path- way components, including Notch, Delta, Smate, master- mind, E(@), deltex, and Su(H) and Hairless (BRAND and CAMPOS-ORTEGA 1990; XU et al. 1990; FORTINI and AR- TAVANIS-TSAKONAS 1994; HING et al. 1994). As all genes thus far implicated in this pathway dominantly modify Notch mutations (V&SIN et al. 1985; DE LA CONCHA et al. 1988; FLEMING et al. 1990; XU et al. 1990; FORTINI and ARTAVANIS-TSAKONAS 1994), we performed genetic screens to find additional components of the signaling cascade. Taking advantage of the phenotype produced by regionally expressed truncated, activated forms of Notch, we screened for second-site dominant modifiers. Ectopic expression of the activated receptor in the eye under the control of the sevenless promoter offers a suitable phenotype for such screens and allows the iden- tification of both enhancers and suppressors.

One puzzling aspect of the activated Notch eye phe- notype is the apparent transformation of the R7 precur- sor into a nonneuronal cell type (FORTINI et al. 1993), since elimination of Notch activity in this tissue can also cause loss of the R7 cell (CAGAN and READY 1989). In the case of the activated Notch phenotype, a develop mental analysis has shown that induction of the R7 cell by the R8 founder cell of the ommatidium is blocked by high level expression of activated Notch driven by the sevenless gene promoter. Due to the use of the heter- ologous promoter, this effect occurs several ommatidial columns behind the morphogenetic furrow of the de-

veloping eye imaginal disc and produces a similar cellu- lar phenotype in all ommatidia within the transgenic retina (FORTINI et al. 1993). In contrast, elimination of Notch activity was accomplished by administering timed heat pulses to flies bearing the conditional tempera- ture-sensitive Notchb' allele and produces a more vari- able phenotype involving all retinal cell types. Indeed, CAGAN and READY (1989) demonstrated that within the eye of a single Notch*l fly, ommatidia with up to six extra photoreceptors are found near other ommatidia lacking the R7 cell (see Figure 5 of CAGAN and READY 1989). These authors showed further that removal of Notch activity suppresses normal cell divisions and sug- gested that when the incorrect number of neural pre- cursor cells are specified, the R7 cell is preferentially lost simply because it is the last photoreceptor cell to be recruited into the ommatidial cluster (CAGAN and READY 1989). This interpretation of the Notchbz pheno- type agrees well with other studies implicating Notch in the allocation of neural precursor cell groups within or immediately behind the morphogenetic furrow (BAKER et al. 1990; BAKER and ZITRON 1995; PARIS et al. 1995). Thus, early removal of Notch activity can lead to loss of the R7 cell by interfering with the initial stages of ommatidial cluster formation, whereas the loss of the R7 cell caused by activated Notch reflects a later involvement of Notch in the inductive interaction be- tween the R8 cell and the true R7 precursor cell within an otherwise normally constructed cluster.

The phenotype we used as the starting point of these screens is caused by the ectopic expression of the trun- cated Notch protein and therefore one could question the effectiveness of this tool in identifjmg components of the Notch pathway. However, the recovery of known components of the Notch signaling cascade seems to validate this approach. Several known components of the pathway were recovered encoding cell surface, cyto- plasmic and nuclear proteins. Both gain-of-function and loss-of-function alleles of N, Su(H), and mum were recovered. In each case, the gain-of-function alleles had the opposite phenotypic effect on activated Notch as the loss-of-function alleles. This is the second screen in which Su(H) alleles were isolated based on their ability to modify Notch phenotypes (FORTINI and ARTAVANIS- TSAKONAS 1994), and the only one in which both loss- of-function and gain-of-function alleles of several loci were found to have phenotypic interactions with Notch. We did not recover any new Ser, E(spl), or H alleles, all of which are thought to be components of the Notch pathway. The absence of new Seralleles is not surprising, as Seris not expressed in the eye, nor do mosaic clones in the eye have any phenotype (X. SUN and S. ARTA- VANIS-TSAKONAS, unpublished results; R. FLEMING, per- sonal communication). Similarly, we did not expect to recover any new mutations in the bHLH genes of the E(@) complex since they have been shown to be func- tionally redundant (e.g., DELIDAKIS et al. 1991). We did

1138 E. M. Verheyen et al.

not, however, recover any alleles of groucho, another member of the E(sp1) complex, or H even though they have been shown to be essential and to act during eye development (DIETRICH and CAMPOS-ORTEGA 1984; FISCHER-VIZE et al. 1992).

In examining the interaction of known Notch pathway mutations with the sa-Nact expressing flies, we found that genetic background plays an important role. Pilot crosses of sa-Notch"', to several known Notch pathway mutations, i.e., Dl, dx, Su(H), gave ambiguous interac- tions due to differences in genetic background among the stocks tested (M. E. FORTINI and S. ARTAVANIS-TSAKO- NAS, unpublished observations). The screens described here permit the examination of modifiers within a con- trolled genetic context.

Apart from Notch pathway elements and novel com- plementation groups identified through the modifier screen, we have recovered a number of mutations in known genes. The known genes fell into two categories. The first class includes genes required for normal eye development, namely Star, E@, pnt, Sos, gl, stg, and sca. Each of these enhanced the phenotype of activated Notch. A number of these genes act in other signal transduction pathways that often function in the same tissues and at the same developmental times as Notch. Genetic interactions between Notch pathway compo- nents and members of other signaling pathways, includ- ing the EGF-R and Ras signaling pathways, have been previously observed (FORTINI et al. 1993; ROGGE et al. 1995; KARIM et al. 1996). The observed genetic interac- tions may reflect cross-talk or cross-regulation between pathways, which cannot at this point be distinguished by genetic analysis. It is therefore not possible to deter- mine whether the observed effects reflect additive or synergistic interactions. The second class of mutants in known genes includes those that affect expression from the sevenless promoter that is driving activated Notch ex- pression. For example, we isolated suppressor muta- tions that affect the gene encoding TATA-binding pro- tein, a component of the transcriptional machinery. Thus, reduction in levels of transcription can presum- ably reduce the expression of the activated Notch transgene, thereby suppressing the phenotype. Another example of such a class of modifiers may be the kismet alleles recovered in the screen. It is possible that muta- tions in kismet, a gene that may encode a structural component of chromatin, may indirectly affect sa-Nact expression by perturbing normal chromatin function (KENNISON and TAMKUN 1988; PATTERTON and WOLFFE 1996).

Several of the mutations defining novel complemen- tation groups recovered as sev-Nact modifiers were noted to independently possess phenotypes in tissues other than the eye such as the bristle, ovary and wing where Notch activity is known to be critical. Phenotypic interactions between other components of the Notch pathway and these novel genes were mild or moderate.

Further molecular and phenotypic characterization will determine whether any of these genes define novel ele- ments of the Notch pathway or whether they reflect a more indirect role in modulating Notch signaling.

The fact that we recovered only a single allele of deltex, a known member of the Notch pathway, suggests that the screens carried out may not have saturated the genome for modifiers of the sev-Nact eye phenotype despite screening >130,000 mutagenized chromo- somes. This may not be an accurate criterion for satura- tion since the reduced fertility and viability of deltex loss- of-function mutations (XU et al. 1990) may have led to an underrepresentation of d x among the mutants recovered.

We carried out a number of genetic analyses to clas- sify the modifiers recovered in the screen. A useful test for involvement in Notch signaling has been the ability of a second site mutation to rescue the negative comple- mentation of certain A x combinations (FOSTER 1975; PORTIN 1975; XU et al. 1990; Table 4). Specific muta- tions in five members of the pathway have been shown to rescue this lethality. These five loci are as follows: N, Dl, mam, d x and S u ( H ) (XU et al. 1990; FORTINI and ARTAVANIS-TSAKONAS 1994). However, the nature of the rescuing mutation is critical. Only gain-of-function al- leles of S u ( H ) can rescue while the more prevalent loss- of-function alleles have no effect. As shown in this pa- per, a putative gain-of-function allele of mum cannot rescue while the loss-of-function mum mutations can (XU et al. 1990). Also, no H alleles have been capable of rescue although Hairless is a component of Notch signaling and is capable of binding directly to the Su(H) protein (BROU et al. 1994). So, while this test would have been very useful in determining if a known or novel complementation group was involved directly in the Notch pathway, it unfortunately could not be used to eliminate any of the loci.

It is important to consider the result of these screens in the context of what is currently known about Notch signaling. Genetic and molecular evidence has led to hypotheses regarding how extracellular signals are transmitted to the nucleus via the Notch receptor as well as raised the possibility that the pathway may be autoregulated by feedback. The current evidence sug- gests that signaling is initiated when the Notch receptor binds its ligand, either Delta or Serrate, through the extracellular EGF-like repeats (FEHON et al. 1990; REBAY et al. 1991). This ligand binding, which may result in receptor multimerization, is positively regulated by the interaction between the Notch ankyrin repeats and the cytoplasmic protein deltex (MATSUNO et al. 1995). The transcriptional induction of the Enhancer of split genes and most likely other downstream targets of Notch sig- naling depends on Su(H), although the mechanism by which Notch activates Su(H) is unknown (BAILEY and POSAKONY 1995; LECOURTOIS and SCHWEISGUTH 1995; SCHWEISGUTH 1995). Su(H) has been shown to interact

Modifiers of Activated Notch 1139

TABLE 4

Rescue of the lethality of Ax negative complementation

Gene Nature of mutation A x ~ ~ ~ / K */+ Ax~~' /Ax"; */ t mam"' Loss-of-function mamsAX8 Gain-of-function Su(H)""'O Loss-of-function Su(H)"'"" Gain-of-function

Loss-of-function ~ l f A K 2

wrpb:p3 Loss-of-function sos"v6 Loss-of-function

Loss-of-function Loss-of-function

~ " 4 G 5

kis?BAS 101aeAK4 Loss-of-function P n f T f 3 Loss-of-function

47 44 28 61 32

21 38 55 67 60 71

34 0 0

41 26

0 0 3 4 1 1

snaggldDX' Unknown 38 0 sAK9 Unknown 41 1 eEM2 Unknown 49 3 warthog*S' Unknown 26 0

The ability of modifier mutations to rescue the lethality of negative complementation between Ax alleles was determined. Negative complementation results in trans-heterozygote female lethality (Ax9"/Ax"). Rescue is assayed by survival of females carrying a second-site mutation ( A x ~ ' ~ / A x " ; */+).

molecularly with the Notch receptor and this interac- tion can be affected by ligand binding (FORTINI and ARTAVANIS-TSAKONAS 1994). The ability of Su(H) to bind DNA can be repressed by Hairless, which is also genetically an antagonist of Notch signaling (BROU et al. 1994). Finally, while every modifier screen involving Notch pathway elements has recovered mastermind mu- tations, its molecular role in the pathway is unknown ( BETTLER et al. 1996).

If the relationship of all the pathway elements is lin- ear as described above, then we would expect loss-of- function mutations in members of the signaling path- way to suppress sm-Nact by reducing the level of signal- ing. For example, a reduction of the ligand Delta would be expected to suppress the sm-Nact phenotype by low- ering signaling through endogenous Notch. However, loss-of-function mutations of Dl as well as N , mum, and dx act as enhancers of the phenotype caused by sm-Nact while gain-of-function mutations of Notch and mum act as suppressors of sm-Nact. This apparent contradiction between expected and recovered phenotypes may be explained by postulating the presence of a feedback loop within the cell expressing sm-Nact.

Indeed, genetic analyses involving Notch and Delta mu- tant mosaics in Drosophila first postulated the existence of a feedback mechanism that eventually regulates the copy number of Notch and Delta molecules in neigh- boring cells (HEITZLER and SIMPSON 1991; HEITZLER et al. 1996). Expression analyses of lin-12 and lag-2, the Caenwhabditis elegans equivalents of Notch and Delta, re- spectively, have provided support for the existence of such an autoregulatory loop (WILKINSON et al. 1994).

In the screens described here activated Notch is ex- pressed under the control of the seuenless promoter.

Therefore, we would not expect that the transgene would be subject to feedback loops that target the en- dogenous Notch promoter. When one copy of Notch is mutant, the ratio of activated us. endogenous Notch changes, consequently feedback regulation could be af- fected leading to an enhanced phenotype. Similarly, reducing deltex, which normally acts as a positive regula- tor of Notch, could perturb the ratio of activated us. endogenous Notch signal leading to enhancement. Loss-of-function Delta mutations that enhance the s a - Nact phenotype may also do so by interfering with nor- mal feedback mechanisms. If a normal consequence of Notch signaling is the downregulation of Delta within the receiving cell, then lowering the dose of Delta by mutation may cause that cell to prematurely respond to constitutive Notch signaling, thus perturbing normal differentiation and producing an enhanced phenotype. Unlike the other genes in the pathway, the results ob- tained for Su(H) are consistent with a role in stimulating downstream effectors of Notch. Loss-of-function muta- tions that reduce levels of Su(H) cause a reduction in signaling from activated Notch, thereby suppressing the phenotype. Gain-of-function Su(H) alleles enhance the rough eye by leading to even greater levels of Notch signaling in the presence of sm-Nact.

In conclusion, the screens described here have led to the identification of genes that are capable of modu- lating Notch signaling. It is clear that these screens are capable of identifylng known pathway elements and therefore it is possible that novel elements linked to Notch signaling may be among the mutants recovered. In addition the recovery of known elements of other signaling pathways including the EGF receptor, pointed, Starand Sos suggests cross-talk between Notch and other

1140 E. M. Verheyen et nl.

pathways. In this regard we note that Notch has been documented to modulate both Ras as well as wingless signaling (HING et al. 1994; ROGGE et al. 1995; AXELROD et al. 1996). Finally, the genetic behavior of the Notch pathway elements, as revealed by their interaction with the constitutively activated form of the Notch receptor in the developing eye, supports the notion that feed- back loops may be an integral part of regulating the activity of Notch pathway elements.

We thank I. REBAY for her participation in the initial pilot stages of the screen. We are grateful to K. MATIHEWS and the Bloomington Drosophila Stock Center and the Umea Stock Center for providing numerous fly stocks. We thank A. SHALET for his help with mutant characterization. We also want to thank D. WASSARMAN and I. REBAY

for providing fly stocks, X. SUN for guidance with the eye sectioning, P. XIA for analysis of pointed, B. PIEKOS for scanning electron micros- copy assistance and J. ROOTE for mapping help. E.M.V. was supported by postdoctoral fellowships from the Howard Hughes Medical Insti- tute and the Patrick and Catherine Weldon Donaghue Medical Re- search Foundation. K.J.P. was supported by the Medical Scientist Training Program grant. M.E.F. was supported by the Helen Hay Whitney Foundation and the Anna Fuller Fund. SA.-T. was supported by the Howard Hughes Medical Institute and by National Institutes of Health Grant NS-26084.

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Communicating editor: V. G. FINNERTY