Interactions are described between the Notch locus of Drosophila melanogaster, and two other loci, scabrous and vestigial, which respectively affect the eyes ...
Copyright 0 1990 by the Genetics Societyof America
Interactions of vestigial and scabrous With the Notch Locus of Drosophila melanogaster Leonard Rabinow and James A. Birchler The Biological Laboratories, Harvard University, Cambridge, Massachusetts 02138 Manuscript received September 29, 1989 Accepted for publication January 1 1, 1990 ABSTRACT Interactions are described between the Notch locus of Drosophila melanogaster, and two other loci, scabrous and vestigial, which respectively affect the eyes and wings. The Notch locus is responsible for mediating decisions of cell fate throughout development in many different tissues. Mutations and duplications of vestigial and scabrous alter the severity of phenotypes associated with Notch mutations and duplications in a manner that is essentially tissue- and allele-specific. These interactions indicate that theproducts of vestigial and scabrous act in conjunction with Notch to stimulate the differentiation of specific celltypes.
similar pleiotropic defects during embryonic or pupal HE product of the Notch locus of Drosophila, ( 1 development (DE LA CONCHA et al. 1988; DIETRICH 3.0, 3C7), affects determination of cell fate and CAMPOS-ORTEGA 1984; SHEPARD,BROVERMAN throughout development, and in a large number of and different tissues (CAGAN andREADY1989; LINDSLEY and MUSKAVITCH 1989; VASSIN,VIELMETTER CAMPO-ORTEGA 1985). These interactions are sensiand GRELL 1968; SHELLENBERGER and MOHLER1975; for a recent review, see ARTAVANIS-TSAKONAS 1988).tive tothedosage of the various componentgene products, indicating stoichiometric relationships must Homozygosity or hemizygosity for Notch null mutaexist among them (CAMPOS-ORTEGA 1988). Among tions ( N ) ,are correlated with embryonic lethality and these interactingloci, Delta is homologous to EGF and hypertrophy of the nervous system at the expense of Notch itself (KOPCZYNSKIet al. 1 988), and a transcript ectoderm. Heterozygosity for null alleles generally from the Enhancer of split is homologous to the Pleads to “notched”wings, from which the locus derives subunit of transducin, a mammalian G-protein (HARits name. Recessive Notch alleles fall intodifferent TLEY, PREISSand ARTAVANIS-TSAKONAS 1988). Other classeswhich complementone another,depending interacting loci are of interest because they might also upon whether the eye or wing of the adult is affected be involved in various aspects of cell differentiation. (LINDSLEY and GRELL1968), implying that the locus In this paper, a novel set of interactions involving contains independent domains specific for tissue deNotch and vestigial and scabrous are described. termination events. The DNA sequence of the Notch Two tightly linked autosomal loci, vestigzal (vg: 2locus reveals similarities to other proteins implicated 67.0; 49DE), and scabrous(sca: 2-66.7; 49D), are in intercellular communication, notably mammalian specific intheir effects on wings and eyes, respectively, epidermal growth factor (EGF) (KELLEY et al. 1987; adult structures which can be similarly affected by KIDD, KELLEY and YOUNG 1986; WHARTONet al. different classes of Notch alleles. Mutations at vestigial 1985)and lin-12 of Caenorhabditis(GREENWALD reduce wings and halteres, give erect post-scutellar 1985; YOCHEM, WESTONandGREENWALD1988), bristles and arecell autonomous (LINDSLEY and GRELL which also affects decisions of cell fate. The Notch 1968). Their phenotype is correlated with cell death protein is localized on the cell membrane (KIDD et al. in the imaginal wingdiscs (BOWNES and ROBERTS 1989) and possesses a hydrophobic region character1981; FRISTROM1969;O’BROCHTA and BRYANT istic of integraltransmembraneproteins. The se1983). Alleles of vg are generally recessive, although quence of point mutations with diverse phenotypes reveals amino acid substitutions in different EGF-like two dominant alleles are known, which are due to repeats of the protein (HARTLEY, and BELL XU and ARTAVANIS- inversion breakpoints in the gene (WILLIAMS TSAKONAS 1987; KELLEYet al. 1987). This finding is 1988). Extensive mutageneses have failed to yield any consistent with the idea that different repeats function recessive vg lethal alleles (LASKO and PARDUE 1988). independently to effect the differentiation of distinct Additionally, although some vg alleles reside on chrotissues. mosomes that carry recessive lethals, such alleles are viable over a deficiency for the region, strongly sugPhenotypes due toNotch nutations aremodified by mutations in a number of other loci which result in gesting that the locus is not mutable to a recessive
T
Genetics 125: 4 1 - 5 0 ( M a y , 1990)
and L. Rabinow
42
lethal form (JIM WILLIAMSand JOHN BELL, persona] communication). Mutations in scabrous result in rough, bulging eyes, occasional duplication of scutellar bristles, doubling of the acrostichal hairs on the notum, and areviable as homozygotes (LINDSLEY and GRELL 1968). We have foundthat vg and sca exhibit dosagedependent interactions with Notch. Alleles of vestigial interact only with Notch alleles affecting the wings, and scabrous alleles primarily, though not exclusively, with Notch alleles affecting the eyes and bristles. These results reveal that genes other than the neurogenic loci can interact in stoichiometric relationships with Notch to affect the differentiationof specific cell types. MATERIALS AND METHODS Stocks carrying vestigial alleles were provided by JOHN BELL,and are described in WILLIAMSand BELL (1988) and WILLIAMS,PAPPU andBELL (1 988). Stocks of Notch alleles were provided by MICHAELYOUNG and the Bowling Green (GRIMStock Center and have been previously described WADE et al. 1985; HARTLEY, PREISS and ARTAVANIS-TSAKONAS 1988; KELLEY et al. 1987; KIDD, LOCKETTand YOUNG 1983; KIDD and YOUNG 1986; LINDSLEY and GRELL 1968; MARKOPOULOU,WELSHONSand ARTAVANIS-TSAKONAS 1989; WHARTONet al. 1985). Alleles of scabrous were provided by the Bowling Green Stock Center andWILLIAM WELSHONS. The translocation stock permitting construction of a segmentaltrisomyfor the scabrous-vestigial region, T(Y:2) G44, was described in the report of theSeattle Drosophila group in Drosophila Information Service,47, supplement, 1971, and was obtained from the Cal Tech Stock sca and Center (now at Indiana University). Deficiencies for vg were obtained from CHAO-TING WU. Drosophila crosses were performed at 25". Interactions among the mutations were testedby crossing Notch mutant fernales with either vg or sca homozygous males.The specific alleles of these three loci examined are listed in Table 1, which summarizes the results of over 250 crosses made in generating vg and sca heterozygotes and homozygotes in Notch mutantbackgrounds. Notch mutantfemaleswere either homozygous for recessive Notch alleles, or heterozygous for a null (recessivelethal) Notch alleleandan X chromosome balancer (FM3 or F M 7 ) . In some cases, heterozygous mutant stocks of ug or sca carried over a balancer (Cy0 or SM6a), wereused,to ensurecomparison of the appropriate genotypes. F, males hemizygous for Notch recessive alleles and heterozygous for vg or sca were scored for enhancementor suppression ofNotch and eithervestigial or scabrous phenotypes. In the case of Notch null mutations, females heterozygous for Notch recessive lethals and vg or sca were similarly scored. Notch alleles in combination with homozygous vg or sca mutations were constructedby crossing doubly heterozygous femalesof the genotype N/+; ug or sca/+ with sibling males which either carried an X chromosome balancer or were hemizygous for a viable allele of Notch and were vg or sca/+. Unlessspecifically noted, all phenotypes reported below were completely penetrant, although varying slightly in expressivity.
RESULTS
The phenotypes of Notch alleles affecting wing margins are enhanced in severity by vestigial and
J. A. Birchler
scabrous mutations: During the course of other experiments, interactions were discovered between mutations at Notch, and deficiencies which included vestigial and scabrous. We analyzed these interactions by combining mutant alleles and duplications of these three loci, thus varying the relative doses of their active products. T h e results of over 250 crosses are summarized in Table 1, and reveal the existence of dosage-sensitive interactions amongNotch, vg and sca. Specific alleles and chromosomal aberrations used in construction of the variousgenotypes are listed in Table1, while the results are described in more generalterms below. Certainexampleshavebeen selected for explicit description for illustrative purposes. Heterozygous mutations in vestigial in combination with Notch alleles affecting wing margins (either heterozygous for recessive lethal Notch alleles, or hemizygous for viable ones), lead to a phenotype that is not predicted from simple additivity of the two mutant phenotypes, demonstrating the existence of synergistic interactions between mutations in the two loci (Table 1, Figure 1). Heterozygosity for recessive vg alleles does not produce a mutant phenotype in an otherwise wild-type genetic background, leadingto at most occasional wing-nicking, depending on theallele used (LINDSLEY andGRELL1968). Such heterozygotes, when combined with Notch nulls/+ in females ( I ( I ) N 2 ;Figure IC) andhypomorphic recessive mutations affecting wing margins such as notchoid (nd; Figure lb) andfacet-notchoid (fa""; not shown), tested in both hemizygous males and homozygous females, respond similarly (Figure 1,f and j). Enhancement of the phenotype of affected hypomorphic Notch alleles varies as a function ofthe strength of the thevg allele used; stronger vg alleles have more dramatic effects (Figure 1, f and g). Notch alleles with effects restricted to tissues other than the wings do not interact with any vg allele tested (Table 1). Since phenotypes associated with Notch alleles affecting the wing are dramatically increasedin severity by heterozygosity for mutations in vg (or vice versa), we tested the effects of further reducing the amount of ug function, via homozygosity for different vg alleles. Increased loss of wing structure is observed in hypomorphic vg homozygotes when combined with mutant Notch alleles that interact with vg heterozygotes. This is illustrated by the weak hypomorph, ug79d5,which does not yield complete wing loss as a homozygote(Figure li),except when the level of Notch function is reduced by heterozygosity for either of the null alleles tested ( 1 ( I ) N 2 ,Figure lj; N@", not shown), or a homozygous strong hypomorphic allele (fa"", not shown). As in the case of heterozygous vg mutations described above,Notch alleles altering adult structures other than the wings do not interact, and no reduction in viability is observed in any double mutant combination of N and vg alleles (Table 1).
Notch Interactions
Mutations in sca do not affect the wing in an otherwise wild-type background, and it was thus surprising to find that interactions also occur between sca alleles and thesame series of Notch alleles that interact with vg mutations. Heterozygosity for either of the Notch nulls tested causes sca heterozygotes to become partially dominant, as evidenced by slight eye roughening (notshown). In N/+; scalsca flies, wing notching is unaffected, but increased scutellar bristle twinning and disruption of notal microchaetae rows are observed. Although heterozygosity for sca alleles has no effect on wing notching associated with nd or fa"", homozygosity for sca mutations further reduces the wing structure in the presence of these two Notch alleles (Figure le), and disrupts rows of notal microchaetae, as observed in N/+; scalsca flies. Again, no lethal interactions were observed in double mutant combinations of Notch and sca alleles. In order to test the specificity of the interactions between vg and Notch, alleles of two other loci affecting wing development, rudimentary (@') and cut (&, Etk, ct"' and ct"), were also tested in combination with heterozygosity for u p , with no interactions found. ctk is particularly sensitive to modification (RUTLEDGE et al. 1988; J. JACK, personalcommunication), and was further tested in homozygotes of vgBGas well as vg79d5. N o interactions were observed in either of these cases. A Notch allele affecting eye morphology interacts with scabrous: The phenotype offacet-glossy,fag, which affects eye morphology, results from misrouting of primary pigmentcells to the fate of secondary pigment cells (CAGAN and READY 1989). This allele is due to the insertion of the flea transposable element (KIDD and YOUNG 1986; MARKOPOULOU,WELSHONS and ARTAVANIS-TSAKONAS 1989), and is altered in phenotype by sca mutations (Table 1). In homozygousfag; sca mutants,approximately 10% of the flies have blackened, necrotic patches of tissue varying in size. Flieswith this phenotype are invariably affected in both eyes, with blackened patches roughly equivalent in size, althoughtheamount of blackening varies substantially among individuals. Mutations in Notch EGF-like repeats interact with vestigial and scabrous: The split (spl), allele of Notch reduces the numberof eye facets, disorders them, and occasionally twins or removes scutellar bristles (CAGAN and READY 1989; LINDSLEY and GRELL 1968). This allele is correlated with an amino acid substitution in the 14th EGF-like repeat of the Notch product (HARTLEY, X U and ARTAVANIS-TSAKONAS 1987; KELLEYet al. 1987). The spl eye phenotype is strongly suppressed in sca heterozygotes, while sca homozygotes show only theroughened eye typical of sca alleles (Figure 2a). The spl bristle component is unaffected. Additional evidence of interactions between Notch and scu was supplied by three mutations isolated as suppressors of spl, and mapped to 2R by WILLIAMWEL-
43
(personal communication). They were generously provided to us, and determined to be scabrous alleles, based on their failure to complement either sca or S C ~ ~ ~ as ' ~ well ' , as their eye phenotype when homozygous (LINDSLEY and GRELL 1968). In contrast, spl is unaffected by any of the eight vg alleles listed in Table 1,which were tested both as hetero- and homozygotes. In addition to spl, Abruptex alleles of Notch are also point mutations in the EGF-like repeats (HARTLEY, Xu and ARTAVANIS-TSAKONAS1987; KELLEYet al. 1987). We tested two of them for interactionswith vg and sca. The first, AxE2, affects wing venation. Its phenotype is unaffected by alleles of vg and sca when heterozygous or homozygous. However, when combined with deficiencies encompassing sca and vg (Df(2R)vg"), or just vg (Df(2R)vg"), wing-scalloping and increased venation defects are observed (Table 1). A second allele, is a recessive lethal and removes virtually all the macro- and microchaetae on the thorax. Its phenotype is reduced in severity by heterozygosity for mutations at either vg or sca. Even stronger suppression of the phenotype is observed in vg or sca homozygotes, which have all four scutellar bristles. Additionally, in scalsca flies, notal microchaetae are wild type in number and pattern. Interactions among duplicationsof the wild-type alleles of Notch, scabrous and vestigial: Since mutations in Notch, vg and sca interact with each other, suggesting dosage-dependent interactions among these loci, we examined the possibility that duplications of the wild-type alleles would have the opposite effects. An insertional translocation of second chromosome material into the Y chromosome, T(Y:2)G44 (44C; 50B), includes the sca and vg wild-type loci, and has no mutant phenotypes associated with Notch, sca or vg in segmental trisomics in an otherwise wild-type background. Such segmental trisomics can be constructed by crossing males of a stock carrying this Y chromosomeinsertion, the corresponding second chromosome deficiency, an attached XY chromosome (In(I ) E N ) ,and a second chromosome balancer ( S M 6 ) , with euploid females. Segmental trisomics are male progeny (T(Y:2)),carrying the Cy marker from the SM6 balancer and the normal second chromosome fromtheirmothers.Fora full description of this system, thereader is referredto LINDSLEY et al. (1972). Accordingly, T(Y:2)G44 males were crossed to selected Notch alleles tested above, and the phenotypes of trisomic F1 progeny were compared to their diploid siblings (Table 1). We found that the severity of the nd phenotype is reduced in trisomics for the sca-vg region (Figure 1h). Extra copies of sea+ and vg+ therefore partially compensate forlesions inNotch. If a mutantallele of either vg or sca is present in one copy of the segmental trisomic, no suppression is observed. This verifies that SHONS
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$0 nE '=,ggpua m3 The duplication of Notch+ in D p ( 1 : I ) Co, results in - c0 L ua "u- x x 2 ;. ,g i? wing-venation defects (LINDSLEY and GRELL1968) * y :% 3 2 r N O M S ..e$ s X" t (Figure Id). These defects are slightly increased in ,uugu .u 0>, , " .c 2 homozygous or hemizygous Dp( 1:1) Co by heterozy2 .c z gr x$ gc zoCugCgj g$ r% , C + g $ pw $ b1 gosity for mutant alleles of ug (Figure ll), and homTI i C " Wkb.8 u .2 " 2 E C .- 5 3 ozyosity formutant sca alleles (not shown). Addi;$; O F tionally, two scutellar bristles are lost in 50-75% of s x m L o m i w 0 a homozygous or hemizygous D p ( l : I ) Co; sca homozy- , & b % a 5 x w ? 2 ,u 0 -sc)o u MI" 2 %: 3 u . E 0 0 ,c x $ u gous individuals, aphenotypenot associated with 43 5 -0 -0c ec 2 ~ ~ 5 0 2;;r c u either Dp( 1:1) Co or sca mutations alone. As might be a E=.:: C, c, z = % 1 $ S u w expected, the phenotype associated with 2 o Dp(1:1) Co is 2 5 ;;; C ' Z E -1 i l i u m 0 8 s ;, suppressed by the segmental trisomic generated by :c s,?u % 62 2 2 - '0 5 c 5 w 5. 'C- T s T(Y:2) G 4 4 , which restores balance to the relative a fj i ' &u +"0-iE;s~ w 9 mc G proportions of these loci and presumably their prod42 6 2 $ ' & a , h $ C Em&..SZ z" 8 ",6' !$sn" C u 0 . a2 s L ucts. Conversely, the phenotype of the weak vg hyS%%.,C 5 2 5 g 5 pomorph, ~ g " is~suppressed ~ , in the Dp( 1:1)Co homo.m .0 - E g E 3 u . 5 2 % ro 2 w a E E . zygotes or hemizygotes, with reduced wing-notching u s f g g m u 3 ~ 0 E a w 0 z.-p '=P.g-, observed. In rare individuals (ca. l%),one or both G u FJc 0 $ 94 - P c , 2 : %s 6 0 wings are completely wild type (Figure lk), which -fj * =-c c *sw:F% 2.2 z indicates that excess Notch product can partially comE %S @ z m - c s 8 ug"; D ~ ( Z Rvg"), ) or only v g ( ~ f ( 2vg"), ~ ) were comz e b 2 u s t;'bined with the Notch alleles tested in Table 1. We z$ 2z 2~0S -3Z: cbk.22 z..:~D ;c0 5,O ?S xe ~~nL o , o % ~ , 0 9~ .O~. h;5; Zu Efound thatthese deficiencies behave simply as extreme u M,; * 'D 5 M,g u 2 & u u ~ u c 2 n l q d m n ") z alleles of vg and sca in combination with all but one 3 c z3m= u -3.5 j gi5 -trr =.c3 o 2.5 Y n 4 of the Notch alleles tested (Table 1). The exception is :+* a a;.5 E Y 3 :. 5 ,5-; z*,g 0,s o2: b o umz fu"", which is an unusual allele in that it is hemi- and 5 2 8-0 - 0 ; M a 9 . 5 g o c C homozygous viable, but lethal when heterozygous .b " * 3 % 5 . g m . , a01 E.8.E 9 U u v M 2 z 3 &!?Q $ 2 , m > u with a Notch null mutation (LINDSLEYand GRELL XJC. J x~~~ sg E$ $ $ P 0 " S 5 ; u 0 b & $ 5 * M3 u E-c >\Lo 1968). The phenotype of fa"" hemizygotes or homo2 : ' B TI I $ + Y&'E%-u2 CE $ S $ & q zygotes was strongly enhanced in ug heterozygotes CI X ;s U L 0 u b,m%22 3i.a and sca homozygotes. Additionally, the rows of notal b .;y3% 2 * 5 b-0 3 cc microchaetae are disturbed in fa"" hemizygotes or A- u u L , ~ - u "z= C g 'E a u * 2% 0 " p*..gu.i C.'z z 2 homozygotes if sca was homozygous mutant, reminiss Ss ymS 3 r0.3- -4 2; k a Y k *C , o z " 1 .-c cent of the interactions observedin fliesheterozygous & 0.0 w . 5 u b - 1 ci -u g 3 0 z a % E 3.k 2.2 c m . 2 z foreither of the two tested Notch null alleles and g * y Z % m 0 iC.$= 'J * m z y s g o y k J a b *z G u Ju yz homozygous for sca alleles. Hemizygousfa""; vg/+ or u .-C L a.a;Q,f ."= u 2rir 2 8 & 5 Loo 0 x:-" E sca/sca are viable, but no hemizygousfu""; vg homo2 3s o w 5 s s 0 .J r e a0 5 E,,, E2,i Ms zygotes or dominant alleles of ug/+ were recovered. z 3 .-F 2LIO.g 0 0 c z c 3 . 5 5 E M; ETz 4 2 2 5.2 Females of these genotypes can berecovered, but 3 z ;.E.$ g; z $ p g.2 5 often have duplicated scutellar bristles. Heterozygotes E G E M3"C1 k ' Z borM for any of the .~ ~ c C: C 5 % % ~ : , ~ deficiencies removing both vg and sca c e +^ zbm0 . 3 . 0 :. g0 y;5.8,Y ( D f ( 2 R )ugc; Dj72R) ugD;D f ( 2 R ) vg"), are inviable in :C0 ; c - c M;;; 2 m 0 zz C i'9.8 2.52 m o + fa"" hemi- or homozygotes, and double heterozygous E c $2 q ~ $ ~ ~ d ~ a &3U u females forfa"" and either vg or sca show wing notchSE.-E%E3b 3 2 kQS2 ing typical of Notch nulls. Dj72R) vg' removes vg+ but O"0
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