Copyright 0 1990 by the Society of America

deltex, a Locus Interacting with the Neurogenic ,Notch, Delta and mastermind in Drosophila melanogaster

Tian Xu and Spyros Artavanis-Tsakonas Howard Hughes Medical Institute, Departments of Cell Biology and Biology, Yale University, New Haven, Connecticut 0651 1 Manuscript receivedJune 1, 1990 Accepted for publication August 2, 1990

ABSTRACT The Notch locus of Drosophila melanogaster, which codes for a transmembrane protein sharing homology with the mammalian epidermal growth factor, is one of a small number of zygotically acting genes, the so called neurogenic loci, which are necessary for the correct segregation of neural from epidermal lineages during embryogenesis. In an attempt to identify geneswhose products may interact with that of Notch, we designed a genetic screen aimed at identifying supressors of certain Notch which are known to affect the extracellular epidermal growth factor homologous domain of Notch. Mutations in two neurogenic loci were identified as suppressors: Delta, whose product was recently shown to interact with Notch and mastermind. In addition, a third, X-linked was shown capable of acting as a suppressor. We show that this gene is the deltex locus, characterize the of deltex mutations, and demonstrate both a maternal and zygotic action of the locus. All deltex alleles behave as recessive viables affecting wing, ocellar and eye morphology. There are allele specific interactions between deltex and various Notch alleles; for example, deltex mutants with a reduced dosage of wild-type Notch die as pupae. deltex also interacts with Delta and mastermind in a fashion that is formally analogousto its interaction with Notch. These results emphasizethe special relationship between Notch, Delta and mastermind suggested by previous work and indicate that deltex is likely to play an important role in the same genetic circuitry withinwhich these three neurogenic loci operate.

N our attemptsto understand the molecular mech- of the neurogenicloci may code for elementsof a cell I anisms underlying the decision of an embryonic interaction mechanismessential for the differentiation cell to choose between a neural or epidermal devel- of various Drosophila tissues (WHARTONet al. 1985; opmental pathway, we have been studying a group of reviewedin ARTAVANIS-TSAKONAS 1988).This hy- Drosophila genes, collectively known as the zygotic pothesis is supported by developmental and genetic neurogenic loci. When mutated to the null state, each studies which suggest that a variety of developmental of the zygotic neurogenic loci, Notch (N),Delta (Dl), events such as the differentiation of the embryonic mastermind (mum),Enhancer of split (E(spl)),neuralized neural ectoderm, ommatidial development or bristle (neu), and big brain (bib), results in a hypertrophied formation, all known to depend on cell interactions, nervous system at the expense of ventral and lateral are also affected by Notch mutations (WELSHONS1965, epidermal structures. This effect is due to the mis- 1971 ; SHELLENBARGERand MOHLER1978; HELD and routing of epidermal precursor cells into a neuronal BRYANT1984; DOEand GOODMAN1985a,b; TECHNAU pathway (POULSON 1937; LEHMANNet al. 1983; JUR- and CAMPOS-ORTEGA1986; TOMLISONand READY GENS et id. 1984; WIESCHAUS et al. 1984; NUSSLEIN- 1987; CACANand READY1989). VOLHARD,WIESCHAUS and KLUDIG1984). Most of the The concept of an involvement of Notch in a cell zygotic neurogenic loci have been shown to be pleio- interactionmechanism impliesseveral interacting tropic affecting both embryonic as well aspostem- components. Indeed, the predicted structures of both bryonic developmental stages (e.g., WELSHONS1965; Delta and (E(@)) are also consistent with an involve- SHELLENBARGERand MOHLER 1978; DIETRICHand ment in cellular interactions(VASSIN et al. 1987; HAR- CAMPOSORTEGA 1984). TLEY,PREISS and ARTAVANIS-TSAKONAS1988; KOP- The molecular analysis of Notch has demonstrated CZYNSKI et al. 1988; PREISS, HARTLEYand ARTAVANIS- that it codes for a transmembrane protein sharing TSAKONAS1988). In an attempt to uncover other homology with the mammalian epidermal growthfac- elements which are involved in the same cell interac- tor (EGF),suggesting that the Notch protein is in- tion mechanism in which Notch participates, a genetic volvedin cell interactions (WHARTONet al. 1985; screen was carried out toidentify genes whose prod- KIDD, KELLYand YOUNG 1986; JOHANSEN,FEHON ucts may interact with Notch protein (XUet al. 1990). and ARTAVANIS-TSAKONAS1989; KIDD et al. 1989). Specifically, we have searched for suppressors of the This ledto the proposition thatNotch and some or all lethality associated with certain heteroallelic combi-

Genetics 146 665-677 (November, 1990) 666 T. Xu and S. Artavanis-Tsakonas

nations of the Abruptex (Ax)alleles (WELSHONS197 1 ; had the new phenotype, thus the position of the new muta- FOSTER1975; PORTIN1975), a group of dominant tion(dXENU) was placed between cu and sn3. Eight recombi- Notch mutationsaffecting postembryonic develop- nants between co and d8Nu(1 y cv dxENu m3/Y and 7 AXE'v fly) were recovered among the 184 y males and 1 16 ment and associated with point mutations in the ex- males. The recombination distance between co and d8L tracellular EGF-like domain of the protein (HARTLEY, was estimated between 2.7 cM (81300) to 3.8 cM (71184). XU and ARTAVANIS-TSAKONAS1987; KELLEY et al. Three AXE2dXENuv fly, three A#' v flyand three AXE2 cu v 1987). This genetic screen led to the identification of fly F2 males were individually mated with y w Ax9''/FM7C independent mutations in two autosomal loci which virgins, and only the crosses involvingAXEZ dXENUv f/Y males produced viable A8'/Ax9'' females. To finally prove that were demonstrated to bealleles of the two neurogenic the deltex is responsible for rescuing the negative loci Delta and mastermind. The results of the screen, complementation between A.?'/Ax9'', an inde endent deltex along with the phenotypicinteraction analyses of mutation (dx) was recombined onto the A rR sn3 chromo- Notch, Delta and mastermind, strongly indicate that some, and shown to be capable of givin viable A8'/Ax9'' females (yA8'dx/FM7C virgins x y w Ax9 '/Y males). two 8 Notch and these loci are integrated in the same The origins of dx, dKSM and dfl: The original deltex allele, genetic circuitry, possibly through direct protein-pro- dx, was isolated by virtue of its recessive wingvein phenotype tein interactions (Xu et al. 1990). Consistent with the which resembles the dominant phenotype of Delta mutations genetic data, recent molecular analysis has indicated on the third chromosome and was therefore named deltex that the gene products of Notch and Delta may be (delta in the X chromosome, MORGAN,STURTEVANT and BRIDGES1922). d2Mwas a spontaneous mutation isoiated in directly interacting via their extracellular domainsand a FZ culture of a cross between a wild-typemale and a that the intermolecular association between these two balancer female carrying a mei-9L mutation, and was kindly proteins is strong enoughto promotecell aggregation provided by ABRAHAMSCHALET. A nonjumper mutation (FEHONet al. 1990). (njp76;S. N. KRISHNAN, personal communication) was also Besides the autosomal Delta and mastermind muta- carried on thesame chromosome. dx" was isolated from the dx" stock by recombining off the SxPb allele from that tions, the genetic screen also recovered twenty three chromosome in the following crosses (GOLUBOVSKY1983; X-linked suppressors. Twenty-one of them are lethal MAINEet al. 1985): a dx" f/FM6 virgin was mated to a y cho Notch alleles, while the other two are viable alleles. In sn3/Y male. The F1 dd'f/y cho sn' heterozygous virgins were this paper we demonstrate that one of the viable X- mated to y cho sn'/Y males. Individual FP males, which had linked suppressors is allelic to deltex, a viable mutation both deltex and singed , were tested for theability to produce homozygous females by crossing to FM6/FM7C previously identified by MORGAN, STURTEVANTand virgins. Two such recombinant males were recovered and BRIDGESin 1922.Phenotypic analyses and comple- gave homozygous females. mentation tests of the existing deltex mutations indi- Thestrains and crosses for complementation tests between cate that they define a single complementation group deltex alleles: y dpNu sn3 virgins x y dXENUsn'/Y, dgMnj"' on the X chromosome.We have further characterized t'v/Y, ec dx/Y or dx" sn3/Y males. AZ' dZNU sn3/FM6 virgins X A#' dXENUsn3/Y, y dXEN" the adult and the maternal effect embryonic pheno- sn3/Y, dXTM njp7' t' v/Y or ec dx/Y males. types associated with these deltex alleles. By examining d2Mnjp7' t' v/FM6 virgins X d2MNJP76 t' v/Y or ec dx/ interactions between deltex and the neurogenic loci, Y males. we show that in addition to Notch, deltex also interacts dx" sn3 virgins x y d8NUsn3/Y males. with Delta and mastermind, thussuggesting a close Thestrains and crosses for the data in Figure 1 were as follows: The crosses involving the farnbmutation were: wa relationship between the productsof these four genes. fanubvirgins X y d.8Nu sn3shY males-FI: y d8NUsn3/wa farnb females x w" farnb/Y males-F2: W' fambd8Nu sn3/Y males X MATERIALS AND METHODS wafanub virgins. The crosses involving the fag mutation were: y dXENUsn3 Strains and crosses: Stocks were maintained and crosses vir ins X fag rb/Y males-FI: y dXENU sn'lf." rb females X y were performed on a standardcornmeal-molasses-yeast-agar drRv" sn3/Ymales-Fz: fag rb dXENusn3/Y males X fag rb virgins. medium containing 0.2% propionic acid or Tegosept as FM6/FM7C virgins X fag rb dXEN" sn'/Y males-F,: FM7C/ mold inhibitors. All cultures were maintained at 25" unless fag rb dPNUsn' virgins X fag rb d3FENUsn3/Y males. specific temperatures were mentioned. Genetic markers and The crosses involvingthe fa"" mutation were: FM7C/w spl strains not specifically mentioned are found in LINDSLEY dXENVsn3 virgins X fa"" rblY males-F1: w spl dgNUsn3/Jano rb and GRELL(1968) or LINDSLEYand ZIMM (1985,1986, virgins x fa"" rb/Y males-F;n: fa"" rb dXENu sn3/Y. 1987, 1990). The crosses involving the spl mutation were: y dXENUsn3 The origin and the meiotic recombination mapping of da?? vir ins X w spl/Y males-F1: y dflN" sn3/w spl females X y The mutation (dpNU)was originally induced in they w Ax9" dgUsn3/Ymales-F2: w spl dXENUsn'/Y males X w $1 virgins. chromosome by ENU (Xu et al. 1990) and thenwas recom- FM6/FM7C virgins X w spl dXENUsn3/Y males-Fl: FM7CI bined onto the A#' sn3 chromosome. It was determined to w spl dfN* sn' virgins X w spl dpNU sn3/Ymales. be proximal to Ax9'' mutation. The meiotic recombination The crossesinvolving the Abruptex mutations were: y position of this mutation was then determined by the follow- dgNusn3 virgins X AXE' sn3/Y males-F1: y dXENUsn31AXE2 sn3 ing crosses: y cu v f virgins were mated to AXE' d.8NU sn3/Y females X y sn3/Y rnales-FZ: A.8'dXENU sn3/Y males X males. The F, y cu v f/A8' d.8Nu sn3 heterozygous females Ax&' sn3 virgins. were then mated toy cv u f/Y males. Three hundred F2 male FM6/FM7C virgins X Az2dXENU sn3/Y males-FI: FM7C/ offspring from the cross were sorted according to their A8' d8*' sn3 virgins X A.8' d3eENU sn3 males. phenotypes. Only one out of 14 of the y cu sn'/Y offspring y AX.' nt vf virgins x ec dx/Y or dXSM njP:t'? 2 v/Y males- deltex Locus and Development 667 A B CD E F GHI

I .I I I .I I --b 3' I IUIU. SP

FIGUREI.-Allelic specific interactions between deltex and Notch loci. The genomic organization of the 40-kb-long Notch locus is schematically shown on the top of the figure: the nine exons, labeled from A to I, give rise to a 10.2-kb mRNA, and are indicated by solid bars connected by lines representing the intronic regions. The various key domains of the corresponding Notch protein are denoted underneath by lines and brackets: SP, signal peptide; EGF-like R, the 36 EGF-like repeats; NR, cysteine-rich repeats present in both Notch and lin-12; TM, transmembrane domain; CDClO-H, five repeats of yeast CDClO homologous sequence (BREEDENand NASMYTH1987); PA, phosphate-binding sequence homology; opa, opa repeat (for more details see WHARTONet al. 1985). The approximate positions (arrows) of the molecular lesions and the phenotypes of different Notch alleles are also depicted. The specific Notch allele of the flies (either homozygous or hemizygous) is given in the top row of the figure, whereas the relevant constitution of the deltex locus is presented in the first column. The observed phenotypes of Notch allele and deltex mutation in the double mutant combinations are presented as: n.c., both Notch allele and deltex phenotypes are present and not changed; S, suppressed; E, enhanced. The particular phenotypes which are enhanced or suppressed are specially indicated after E or S. For example, E(nd2 wing phene) means that only the wing phenotype of ndz is enhanced. Lethal = such double mutant flies are not viable; * = in addition to dZN",d8' and dx were also tested; ** = the wings of these flies are never nicked, but the extra vein materials phenotype is stronger than either or Notch duplication (Cos479) alone. For details of strains and crosses, see MATERIALS AND METHODS.

FI:y AXE2 cu v f/ec dx or y AXE2 cu v f/d2MnjP7' f v females The crosses involvingthe mam'L1'5mutation were: y d8N" X y AX"* cu v f/Y males-F2: y AXEZ dx/Y or y AX"' dgMnjp76 f sn3 or ec dx virgins X cn bw sp mam'L''5/Cy0 males. v/Y sterile males. y dXEN" sn3/FM7C virgins X cn bw sp mam'L1'5/Cy0 males. Fz: y AXE2 cu v fly AX"'dx virgins X FM7CIY males-Fs: y The crosses involving the neu'F65mutation were: y dXENu AXE2 dxlFM7C virgins X ec dx/Y or y sn3/Y males. sn3 or ec dx virgins X neuIF6'/TM3Ser or A?' sn3/Y;neuIF6'/ The crosses involving the nd and nd2 mutations were: w" TMl males. nd or nd2 vir ins X y dXE2 sn3/Y males-F,: wa nd/y dXENu sn3 The crosses involving the biboDo5mutation were: y dXENu or nd2/y d8' sn3 virgins X FM7CIY males-Fz: wa nd dXE2 sn3 or ec dx virgins X cn bw sp biboWS/CyOmales. sn3/FM7C or nd2 dXE2 sn3/FM7C virgins X w" nd/Y, nd2/Y,y The crosses involvingmutations of the E(@) locus were: dx"Nusn3/Y, y W'N"'~/K Cos479/+ or FM7CIY males y dXENusn3or ec dx virgins X l(gr~)~'/TMbB, E(S~L)~~~~/TM~B The crosses involving the Notch deficiency N5419were: y and e4E(~pl)E73 txITM6Bmales. wa ~5419/FM6 virgins X y dXENu sn3/Y males-F,: wa NS4l9 Immunocytochemistry: were dechorionated in dX"Nusn3 virgins X FM7CIY males-Fz: y wa N"''dx""" sn !y / 50% Clorox solution and fixed in a mixture of heptane and FM7C virgins X y dXENu sn3/Y, wa N54'9/Y;Cos479/+ or 4% paraformaldehyde for 30 min at room temperature. FM7CIY males-Fs: y wa N5419 d#' sn3/Y;Cos479/+ males X Vitelline membranes were removed in mass by the heptane- C( 1)RMtwfvirgins. methanol method of MITCHISON and SEDAT(1983). Em- y dXEN sn virgins X y wa N5419 dXENUsn3/Y;Cos479/+males. bryos were washed for 1 hr in BSN (balanced salt solution The crossesinvolving the Notch transformant Cos479 pH 6.95; 0.04 M NaCI, 0.05 M KCI, 0.01 M MgS04, 6 mM were: y dX"NUsn3 or ec dx virgins X y wa N54'9/Y,Cos479/+ CaCl2, 10 mM Tricine, 20 mM glucose, 50 mM sucrose, 0.2% males. bovine serum albumin with 3% normal goat serum, and C( 1 )A, y;Cos479/+ virgins X y dXENusn3/Y or ec dx/Y males. 0.1% saponin) followed by overnight incubations with 1:2 The strains and crosses for the data in Figure 2 were as dilutions of 22C10 and INV4D9 monoclonal antibodies follows: The crossesinvolving the Notch deficiency N5419 (Mab) at 4 O . Washing was performed with phosphate-buff- were: (see strains and crosses for Figure 1). ered saline(PBS) three timesfollowed by an additional The crosses involving the Dl9p39mutation were: y dgN" preincubation for 1 hr inBSN. Horseradish peroxidase sn3 or ec dx virgins X D19p39/TMlmales. (HRP)-conjugated goat anti-mouse immunoglobulin G y dXENUsn'shFM7C virgins X D19p39/TMImales. (Jackson Laboratories) was used at a 1:500 dilution in BSN 668 T. Xu and S. Artavanis-Tsakonas

-dx dx pupal pupa!. Viable, dx Or y I lethal 1 lethal I :;:;** 1 dx dx

-dx + N &dx Dl &dx weakdx wt

FIGURE2.-The genetic interactions between deltex and zygotic neurogenic loci. The effects of mutations at different zygotic neurogenic loci on the deltex flies are summarized. The top row indicates the genetic constitution of the neurogenic locus and the left-most vertical column indicates the genetic constitution of the deltex locus. In the case of the Notch deficiency, only dXENUwas tested. Both dx”NUand dx were tested with the mutations from the rest of zygotic neurogenic loci. The observed phenotypes are presented as: pupal lethal, such flies die as pupae; Viable, dx, such flies are viable and exhibit deltex phenotypes; N & dx, such flies exhibit both Notch and deltex phenotypes; wt, no notable adult mutant phenotype is associated with these flies; * = given the complexity of the E(sp1) region, three different mutations were used in the tests. They were a large deletion l(gro)”, a small deletion E(~pl)”~~~and a point mutation E(~pl)”’~(PREISS, HARTLEY and ARTAVANIS-TSAKONAS1988). ** = about 10-15% of y dXENUS~’/Y;D~~~’~/+ flies and y dflNusn’/Y; cn bw sp mam“”’/+ flies develop into adults. For details of strains and crosses, see MATERIALS AND METHODS. for 2 hr at room temperature followed by washing in PBS. (MORGAN,STURTEVANT and BRIDGES1922). Indeed, HRPreactions were performed in 0.5 mg/ml DAB and genetic complementation tests showed that the sup- 0.015%hydrogen peroxide for 3-5 min. Anti-HRP staining was performed utilizing fluorescein conjugated antiperoxi- pressor is allelic to the deltex (dx) mutation and was dase antibodies (Cappel). Embryos were treated as above. called deltex-ENU (dXENU),reflecting the fact that it Incubations were performed overnight using a 1:500 dilu- was induced by nitrosoethylurea (ENU) (Xu et al. tion, followed by rinsing with PBS. Embryos staining with 1990). 4,6-diamidino-2-phenylindole(DAPI) were treatedas above and incubated with 100 ng/ml DAPI in BSN for 10 min, To test if preexisting deltex alleles were also capable followed by rinsing with PBS. All embryos were mounted of rescuing A#’/AX~~’flies, the original deltex allele, in 2% n-propyl gallate in 70% glycerol. dx, was recombined onto theA$’ chromosome. Since Scanning electron microscopy:Whole flies which either A#’ dx/A~~~’/+flies were foundto be viable (see crawled out of puparia by themselves or were helped out of puparia by dissectionwere desiccated, coated with MATERIALS AND METHODS), the dominant suppression gold-palladium, andviewed with ISISS-40 scanning electron of the Ax negative complementation is not peculiar to microscope. the d8NUallele. These observations wereextended by showing that a different lethal combinationof two Ax RESULTS alleles, namely A#’ and Ax75c24,could also be rescued Mutations in deltex can suppress the negative com- by d$Nu (Ax75c24 +/A#* dflNU). plementation between Abruptex alleles: Recombina- Adult phenotypes of deltex mutations: Besides the tion and complementation analysis of the mutant chro- newly induced mutation dXENu,three additional deltex mosome carrying oneof the viable, X-linked suppres- alleles are available: dx, dXSM and dz.All four muta- sor of the A#’/AxgB’ negativecomplementation, tions behavegenetically as recessive viables and define revealed that this mutation affects a gene other than a single complementation group on the X chromo- Notch. This suppressor has a recessive wing as well as some (see MATERIALS AND METHODS for complemen- an ocellar phenotype(Figure 3, A and B). Meiotic tation crosses). dXSM and dxfNUare both fully penetrant mapping placed this mutation about 3 cM proximal strong alleles. The adult phenotype associated with to crossveinless (cv) on the X chromosome (see MATE- these deltex mutations consists of extra wing vein RIALS AND METHODS). The phenotype and the map material especially at thedistal ends of the wing veins position of this mutant were consistent with it being (deltas), and frequently nicked wing margins and tips an allele of the previously described locus deltex (dx) (Figure 3A). In addition to thewing phenotype, their

FIGURE3.-Phenotypes and phenotypic modifications of deltex mutations. A, The wing of a y dflNu sn3/Ymale: note the notches and extra vein material along the veins. B, The ocellar region of a y dx”N“ sn3/Y male: note the fusion of ocelli with the lack of ocellar bristles. C, The wing of an Afi2 sn3/Y male: note the wing vein gaps at the posterior ends of the fourth andfifth longitudinal veins. D, The wing of an AX“’ dXENU sn3/Y male: the phenotype (wing vein gaps) of the AXE2 mutant is completely suppressed, whereas the deltex phenotypes are strongly enhanced. E, The wing of a nd2 female: shows distal wing notches. F, The wing of a nd2 dZNU sn3/nd2+ + female: a severe wing material loss phenotype is observed in this genetic combination. The w“ nd dflNU sn3/+ nd+ + flies exhibit a similar phenotype. G, The wing of a y dXfi:NUsn’/Y;H/+ male: note that the wing phenotype of deltex is completely suppressed by the Hairless mutation. H, The wing of a y dx””” sn’/Y;Cos479/+ male: note that the wing nicking phenotype of deltex is completely suppressed by a Notch duplication. deltex Locus and Development

C

E

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H 670 T. Xu and S. Artavanis-Tsakonas ocelli are closer or are fused and thehairs and bristles could also be recognized by staining with anti-HRP in the region appear abnormal or are missing (Figure antibody which is knownto label neurons in Drosoph- 3B). The phenotypes of dx are weaker than the pre- ila embryos (JAN and JAN 1982). Although the number vious two, primarily showing onlyextra vein material of the anti-HRP-positive cells varies in such embryos, at the wing tips. Wing notchings and ocelli abnormal- all of them had fewer stained cells than wild-type and, ities occur infrequently. d8is the weakest allele and in addition, the anti-HRP-positive cells appeared dis- adults display only a weak vein phenotype at thewing organized (Figure 5, A and B). This phenotype is tips. clearly different from the neurogenic phenotype of All four deltex mutants have a weak rough eye null mutations of anyzygotic neurogenic locus,in phenotype which is not obvious under the dissecting which hypertrophy of the nervous system is reflected microscope. Scanning electron micrographs revealed by a massive anti-HRP staining pattern. that eye morphology is characterized by infrequently Embryos from the same cross were examined with missing or duplicated bristles as well as an irregular two additional markers. First, they were labeled with array of ommatidia (Figure 4, A and B). The visual monoclonal antibody (Mab) 22C10, an antibody ability of the deltex mutations was tested by subjecting shown to recognize primarily peripheral nerves (Fu- dXENu flies to an edge test(LIPSHITZ and KANKEL JITA et al. 1982). As shown in Figure 5, C and D, it 1985). According to this test the dafNU flies do not seems that the structures recognized by MAb 22C10 show any visual abnormality (data not shown). were alsoaffected in embryos withmutant phenotype. Since some of the deltex flies have spread wings, We consistently saw that MAb 22C10recognized these flieswere subjected toa flight testing assay fewer and apparently disorganized cells in these em- (CHASE1986). The original deltex allele, dx, displays bryos. In order to visualize non-neuronal structures, a temperature sensitive flightless phenotype. Mutant we examined the embryoswith a Mab (INV4D9) flies grown at 25" cannot fly while those grown at whichrecognizes the product of the segmentation 18" behave normally.In contrast, dXEN" and dXSM flies gene engrailed (PATELet al. 1989). In this case abnor- are incapable of flying at both temperatures. Finally, mally developed embryos at germband extension the weakest deltex allele, d8,does not show any flight stage, as well asat older stages, were easily recognized defect in this test. by their staining pattern (Figure 5, E and F). As seen The maternal effect embryonic defects of deltex in Figure 5F, it is clear that these embryos had fewer mutations: Although deltex mutations are viable, we cells expressing engrailed. have found that about 40% of the eggs laid by homo- The data obtained with the three markers suggest zygous dXENUfemales fail to hatch (ydXENu sn'ly d#Nv that tissues in the affected embryos may be degener- sn' virgins X y dpNusn3 males). In contrast, hetero- ating. Staining with DAPI, a dyewhich stains all zygous dflNUfemales (y dZNUsn3/y cho sn' virgins x y nuclei, revealed that 4-5-hr embryos from homozy- dXENUsn' males) laid normal looking eggs, and more gous dXENU females have fewer, irregularly arranged than 99% of the eggs from such a cross hatched, nuclei (Figure 5, G and H). As can be seen in Figure indicating full viability of the homo- or hemizygous 5H, many cells in the have large, abnormally deltex embryos.Respectively, about 45%, 40% and shaped nuclei, suggesting they were undergoing nu- 16% of the eggs laid by homozygous dXSM,dx and d8 clear degeneration. It was also noticed that some of females also failed to hatch (dx" njp76? v/dgMnjp76 the old embryos (25-30 hr at 25")had very few DAPI- ? v, ec dxlec dx or dd' sn'ld8 sn'). Even though the positivecells (data not shown). The DAPI staining exact percentages of unhatched eggs between these pattern therefore is also consistent withthe notion of deltex mutations are not directly comparable since degenerative events. each deltex allele is associated with different genetic Allelespecific interactions between deltex and background, these data indicate that deltex is associ- Notch: The dominant suppression of the negative ated with a maternal embryonic effect. complementation between Abruptex alleles by deltex To further characterize the deltex maternal effect, mutations has already been described. A single copy eggs laid by homozygous dZNufemales (y dZNUsn'ly of a deltex mutation has noapparent effect on a dfiNUsn') were collectedand examined. About 6% of homozygous AXE' fly. For example, the AXE2 the eggs were either obviously shorter than normal d#Nu/A#2 + is phenotypically indistinguishable from eggs or had defective dorsal appendages (data not Ap2/A2'. In contrast, homozygous or hemizygous shown). As revealed by cuticular preparations, the deltex mutations completely suppress the AZ' pheno- majority of the unhatched eggs (eggs that were col- type and at the same time the deltex phenotype appears lected and aged for more than 24 hr at 25") did not to be enhanced by the A8' mutation (Figure 3, C and develop anycuticular structures, whereas a small frac- D). In addition, we observed that deltex,Abruptex tion of them had defective or wild-type looking cuti- double mutations have a pronounced effect on male cles (data not shown). Abnormallydeveloped embryos fertility: hemizygous A#' dZNUmales have low fertil- deltex Locus and Development 67 1

r

FIGURE4.-ThC cyc defects associated with dx'," mt~tantadults and with dead pupae causcd by reducing h'otrh dosage in deltex mutants. A, An eye ofy ddr" sn3 female: note the relatively normal size of the eye. U. An area of the same eye in A: note the duplicated and missing brisltes between facets. C, An eye of y waN'"' dir" sn'/y + + d&"" sn' dead female: note the size of the eye is smaller than wild type. D, An area of the swne eye as in C, showing square facet array which is remincent of an ey" mutant eye (HARTMANand HAYFSI97 I). ity while the hemizygous Ag2 dx (or dP") males are in homozygous, hemizygous, as well as heterozygous, completely sterile. combinations and the results are summarizedin Fig- The relationship between deltex and Notch was ex- ure 1. The top partof the figure is a schematic amined further by undertaking a systematic pheno- representation of the Notch gene. The approximate typic analysis of double mutant combinations between position, in relation to coding regions (dark bars), of dXk;NU and various Notch alleles. To achieve this, Notch the lesions associatedwith each of theNotch mutations mutations affecting different domains of the gene, examined, is indicated anda brief phenotypic descrip- wererecombined onto the chromosome carrying tion of each mutantis also given. dlNu(see MATERIALS AND METHODS). Possible syner- dgNUdoes not show any noticeable interactionwith gistic effects betweenNotch and deltex were monitored farwb,fag or split. These three mutants affect, respec- B

1 a

C D deltex Locus and Development 673 tively, the 5' untranscribed region, the second intron unchanged (y dxEm sn'/Y; H/+Figure 3G). and the 14thEGF-like repeat of the Notch locus (KIDD deltex interacts with neurogenic mutations Notch, and YOUNG 1986; HARTLEY,Xu and ARTAVANIS- Delta and mastermind: Genetic analysishas shown TSAKONAS1987; KELLEYet al. 1987; MARKOPOULOU, that wild-type development is very sensitive to the WELSHONSand Artavanis-Tsakonas 1989; RAMOS et dosage of the Notch locus: a female with three, rather al. 1989), and all three have pronounced effects in than two, copies of Notch has a Conjluens phenotype, the eye (WEISHONS 1965; KEPPY and WELSHONS consisting of irregularly thickened wing veins (WEL- 197,5). Hemizygotes ofdouble mutants of dXEm with SHONS 1965); a female with only one copy of Notch fanub,fa8 or split, display both the deltex and Notch displays wing notching, and flies completely lacking phenotypes with no significantmodification cfa" Notch activity die as embryos displaying the neuro- dXENU/Y,fa" dXENu/Y, or spl dXEm/Y). In contrast, a genic phenotype. We have shown that an extra copy striking interaction is seen between dXEm and thetwo of the Notch gene suppresses the deltex phenotype. We notchoid mutations. Both nd and nd2 have been pre- have further found that the haplo-insufficient behav- viously shown to be caused by mutations affecting the iour of Notch, underlying the dominant adult wing intracellular part of the Notch protein (XUet al. 1990). notching phenotype, also manifests itself inthe inter- The hemizygous double mutants nd dXENUor nd2dXEw action between Notch and deltex. never reach adulthood despite the fact that each of From the crosses of double heterozygous N defi- the single mutants is viable. The lethal period of such ciency and dXEN virgins to hemizygous dXENUmales ( y double mutants has not yet been determined. Fur- w" N5;54I9dXENU sn3/FM7C X y dXENUsn3/Y) or homo- thermore, one copy of the dXENU mutation strongly zygous dXENUvirgins to hemizygous N and dflNumales enhances the notchoid wing phenotype and visa versa carrying a Notch duplication on their third chromo- (nd dpNU/nd +, nd2 dXENU/nd2+, nd dXENu/+ dXENu, some (y dXENUsn3 X y w" NS4l9dXENu sn3/lY; Cos479/+), nd2 dXEw/+dXENu; see Figures 1 and 3, E and F). we noticed that flies homozygous for dXEm and het- Interestingly, a Notch mutation, fa"", which has a sim- erozygous forthe Notch deficiency (W419 dXENU/+ ilar phenotype as notchoid alleles (WELSHONS1965), dXEw) were missing from the progeny. We observed does not show anynotable phenotypic interaction with that one-third of the pupae from the first cross failed dXENu. Hemizygotes of double mutations of fa"" and to eclose. By dissecting the pupal cases, we could not dXENU (f2'dXENu/Y) are viable and display both fa"" detect any obvious morphologicalabnormalities. The and deltex phenotypes. yellow, singed bristle phenotypes, the deltex ocellar The deltex phenotypes were also found to be par- phenotype and the dominant thoracic bristle pheno- tially suppressedby an extracopy of Notch. The wings type associated withNotch were clearly visible in these of dXENu flies which also carry a duplication for the dead pupae, indicating that they represented the miss- Notch locus (Cos479; RAMOS et al. 1989) are never ing class of the cross, i.e. the offspring which were nicked (Figure 3H), and have thickened veins. Since heterozygous for N and homozygous for dXENu. A both deltex as well as supernumery copies of Notch closer examination of the dead offspring heterozygous confer a similar vein phenotype, it is unclear whether for N and homozygous for dXENUrevealed that their the phenotype is contributed by the deltex mutation, eyes are significantly smaller than the wild type (Fig- the extra copy of the Notch gene or both. The ocellar ure 4C). Scanning electron micrographs show that the phenotype of these flies is either completely or par- small eyes bear the infrequent missing or duplication tially suppressed. In addition we also noticed that the bristle phenotype of dx mutants. In addition, the Hairless (H)mutation, which has been shown to inter- ommitidia have a square shape instead of the hexag- act with some of the neurogenic loci (VASSIN, VIEL- onal shape of wild-type eyes. Bristles are seen at each METTER and CAMPOS-ORTEGA 1985), does suppress corner of a square-shaped ommitidium (Figure 4D). the deltex adult phenotypes (wings and ocelli)in a The genetic screen for supressors of the Abruptex dominant fashion while its own phenotype remains negative complementation has shown that in addition

FIGURE5.-The embryoic phenotypes of eggs produced by homozygous d8w females. A, A y d8Nu sn3 embryo which was produced by d8Nuparents shows an anti-HRP-staining pattern that is similar to wild type. B, A y dxENu sn3 embryo from the same cross which was at a similar age as the one in A, and shows abnormal anti-HRP-staining pattern: note there are fewer anti-HRP-stained cells and the stained cells are disorganized. C, A y dSNusn3 embryo which was produced by dgNUparents shows a staining pattern of Mab 22C10 that is similar to wild type. D, A y d#Nu sn' embryo from the same cross which was at a similar age as the one in C, and shows an abnormal MAb 22C10 staining pattern: note theembryo has less MAb 22C10-positive cells than the one in C and, in addition, they are disorganized. E, A y dXENu sn3 embryo which was produced by d8Nuparents shows a staining pattern of MAb INV4D9 that is similarto wild type. F, A y dSNusn' embryo from the same cross which was at a similar age as the one in E, and shows abnormal MAb INV4D9 staining pattern: note there are no MAb INV4D9- positive staining cells in most of the embryo. G, A 4-5-hr-old y dXSw sn' embryo which was produced by dxENUparents shows an abnormal DAPI-staining pattern: note the nuclei are disorganized. H, An area of the embryo in G showing many abnormal nuclei which may undergo degeneration. 6’74 T. Xu and S. Artavanis-Tsakonas to deltex, Delta and mastermind are the other two loci polarity in the embryo, one can argue that the neu- capable of suppressing the negatively complementing rogenicgenes define elements of adevelopmental Abruptex alleles. We have asked whether deltex can pathway, since they display identical mutant pheno- interact with Delta and mastermind as well as with the types. In addition to thezygotic neurogenic loci, there remaining zygotic neurogenic loci by examining the is a considerable number of maternally acting genes phenotypes of doublemutants. The results of this which have been shown, by virtue of specific mutant analysis are summarized in Figure 2. conditions, to confer neurogenic phenotypes (PERRI- No interaction was observed when deltex mutations MON,ENGSTROM and MAHOWALD1989; SCHUPBACH were combined with mutations from the E(spl), bib or and WIESCHAUS1989). Thus, it is clear that the mis- neu loci (see MATERIALS AND METHODS). Interestingly routing of an ectodermal cell into a neural develop- however, we foundthat both mum and Dl behave mental pathway can be influenced by a rather large similarly to Notch: namely, deltex mutants with only number of genes. Conversely, a gene involved in the one wild type copy of Dl or mum can also lead to pupal cell interaction mechanism inwhich Notch partici- lethality (d8Nu/Y;mam’L115/+and d8Nu/Y;D19P39/+). pates, does not necessarily have to exhibit a neuro- However, unlike deltex females carrying one copy of genic phenotype. deltex was identified by virtue of its a Notch deficiency, the lethal phenotype of deltex mu- interaction with a neurogenic locus rather by its neu- tants carrying one copy of Delta or mastermind muta- rogenic phenotype, but the evidence we have gath- tions is not completely penetrant. We found that about ered in the present work suggests that this gene is 10-15% of the d8Nu/Y;mam1L115/+flies and thedflNu/ intimately related to neurogenic gene function. Y;D19p39/+ flies eclosed. In both cases, a small eye deltex was identified during our attempts to dissect phenotype, which is weaker than the one seen in the the genetic circuitry in which the neurogenic locus analogous Notch doublymutant combinations, was Notch is integrated because it is capable of suppressing detected in the dead pupae as well as in the escaped the phenotype of the negatively complementing flies. In addition, flies which are double heterozygous Abruptex alleles (XU et al. 1990). The phenotypic for dx and N,D1 or mum show a weak dominant dx analysis we have carried out has shown that deltex is phenotype. We have not yet determined whether the important for both embryonic and postembryonic de- penetrance of the pupal lethality is dependent on the velopment. The four deltex alleles examined here specific Dl or mum alleles. We also tested the original display similar phenotypes which clearly differ in se- dx mutation which, as previously mentioned, is a weak verity, suggesting that at least some of them are hy- deltex allele, with the zygotic neurogenic mutations pomorphic alleles. None of these mutations displays a except Notch. We found this allele to behave similarly neurogenicphenotype. In this regard, it is worth to d8Nuin terms of interacting with Delta and master- emphasizing that we do not know what the amorphic mind. However the number of escapers in the lethal phenotype of deltex is, since deletions uncovering the combinations between dx and or Dl9p39 is cytogenetic location of deltex do not exist despite the significantly higher than the corresponding combina- many mutagenesis screens involving the X chromo- tions with dxENu. These observations suggest, again, some. The cytological position of the deltex locus was functional links among Notch, Delta and mastermind, previously assigned between the 6A3.4 to 6F10.11 and emphasize the sensitive dosage relationship be- polytene chromosome region (DEMERECet aE. 1942). tween these loci. Using duplications of the region 6C and duplications coveringregions proximal to 6C we have recently DISCUSSION shown that they do not complement any of the deltex A fundamental issue in the analysis of any develop- alleles (unpublished results), placing the deltex locus in mental event deals with the complexity of the genetic the 6A.B region. The finding that the d8mutation circuitry involved in controlling the fate of cells dur- has a P element inserted in 6A is consistent with this ing that event. We are concerned with the question localization (unpublished results). If we can show that of how many genes are part of the logic that governs this P element disrupts the deltex gene, thenimprecise neuroblast differentiation in the neurogenic region. excisions of the element will provide uswith small The genetic screens of NUSSLEIN-VOLHARDand WIES- deletions in the region which, presumably, will be null CHAUS (1 980) for embryoniclethals revealed the ex- alleles of the locus. istence of sixzygotically actinggenes which, in a Even though all four deltex alleles are viable we have mutant state, can confer neurogenic phenotypesOUR- seen that the viability can be dramatically reduced by GENS et al. 1984; WIESCHAUS, NUSSLEIN-VOLLARDand a maternal effect: a certain percentageof the eggs laid JURGENS 1984; NUSSLEIN-VOLHARD, WEISCHAUSand by homozygous deltex mothers died before hatching. KLUDIG1984). By analogy to other groups of genes Examination of the unhatched embryos from deltex which are shown to control specific developmental mothers using several antibodies as specific cellular events, such as the acquisition of anterior-posterior markers as well as a general nuclear marker, indicated and Developmentdeltex Locus and 675 considerable variability ofthe mutant phenotype. The judge from such observation, the lethality ofthe deltex development of these embryos appeared to arrest at notchoid double mutants is not a simple additive effect various embryonic stages, and the staining patterns betweentwo mutations affecting the sametissues. obtained with all the markers revealed considerable Both notchoid alleles were shownto be point mutations cell death and tissue degeneration. affecting the intracellular, carboxy-terminal part of The genetic screen and subsequent analysis of sup- the Notch protein (Xu et al. 1990). The molecular pressors of the pupallethality caused by negative lesion associated withfa"" is not known, but extrapo- complementation between Abruptex alleles indicated a lating from the intragenic meiotic recombination sensitive dosage relationship between Notch and the analysis, it is likely that fa"" affects the extracellular three suppressors Delta, mastermind and deltex. It was portion of Notch (YEDVOBNICKet al. 1985). The ques- found that a negatively complementing mutant com- tion of whether this lethal interaction between deltex bination is rescued by simply lowering the gene dosage and the notchoid mutations reflects direct molecular of Delta or mastermind. The same appears to be true interactions with the intracellular part of Notch must for deltex assuming that the deltex alleles at hand are await the molecular characterization of deltex. When hypomorphic. For instance having one copy, rather considering the specificity ofthe deltex, notchoid inter- than two, of Delta rescues the lethal Abruptex combi- actions, it is worth noting that genetic analyses have nation. The study reported here, involving the inter- revealed that mutations from two neurogenic loci action of deltex mutants and mutations of the neuro- E(sp1) and mum as well as from scabrous and vestigial genic loci revealed sensitive dosage interactions be- also enhance the notchoid phenotype (RABINOWand tween deltex and Notch, Deltaor mastermind. We found BIRCHLER1990; Xu et al. 1990). that the reduction of one dose of Notch,Delta or An additional feature of the deltex phenotype war- mastermind genes in deltex mutants led to a similar rants comment: we have shown that the reduction of phenotype observed between negative complementat- one doseof Notch,Delta or mastermind genes in a ing Ax alleles, namely pupal lethality at similar devel- homozygous deltex background leads to a late pupal opmental stages. lethality. Scanning electron microscopy of the dis- Recent biochemicalwork has suggested a direct sected pupae has shownthat theeyes ofdeltex mutants association between the gene products of Notch and with N deficiency are smaller and have a square facet Delta, which was one of the three suppressor genes array which, we note, is reminiscent of the phenotype recovered by the genetic screen. These experiments of the eyeless mutation, ey (HARTMAN andHAYES showed that the association between Notch and Delta 1971). In ef, the mutant phenotype seemsto be proteins on cell surfaces is strong enough to promote caused by the failure of proper elongation of the the aggregation of these cells and revealed in addition secondary pigment cells along the horizontal axis that the Delta expressing cells are able to form aggre- (READY,HANSON and BENZER1976). The underlying gates between themselvesor with the Notch expressing cause of the phenotype is not known. cells whereas the Notch expressing cells are only ca- Since deltex mutations affect postembryonic stages pable of aggregating with the Delta expressing cells and the embryonic phenotype associated withthem is (FEHONet al. 1990 ). This result suggests that Notch not neurogenic, the relationship of deltex to the de- molecules maycompete with Delta molecules, a notion velopment of the neurogenic region remains unclear. consistent with the dosage sensitive relationship be- Indeed, we do not have any evidencethat deltex plays tween the two genes revealed by genetic studies. Al- a role in neurogenesis and one could argue that the though there is no reason to believe that deltex or documented relationship between deltex and the neu- mastermind encode proteins similar to Notch and Delta rogenic genes is relevant onlypostembryonically. proteins at this point, given the genetic data we ob- Nevertheless, our work has shown that deltex displays serve, it would not be surprising if the molecular dramatic interactions with the neurogenic loci. Not analysis ofdeltex reveals that its gene product interacts only was it found to interact with Notch but it was also directly with all or some of these three neurogenic shown to display strikingly analogousinteractions with loci. the two neurogenic loci Delta and mastermind. In Besides interacting with the Abruptex alleles, deltex addition, deltex mutations behave, vis a vis to Notch, also interacts with other Notch mutations apparently similarly to Dl and mum mutations. Like Dl or mum inan allele specific manner. Flieshomozygous for mutants, deltex alleleswere shown to be dominant dPNUor any of the two notchoid alleles (nd and nd2) supressors of the lethality caused by negative comple- alone are viable. In contrast, double mutant combi- mentation between the Abruptex alleles. nations of dpNuwith any one of the notchoid alleles Interpreting the interactions of deltex with any one are lethal. However, the double mutations between of the neurogenic loci or gaining insight intothe dXENUand fa"", which has a phenotype very similar to biochemical nature of deltex and its role in develop- the notchoid alleles is viable. Thus, as far as one could ment must await the molecular analysis of this locus. 676 T. Xu and S. Artavanis-Tsakonas

The work reported here emphasizes the special rela- HARTMAN,H., and T. L. HAYES,1971 Scanning electron micros- tionship between Notch, Delta and mastermind sug- copy of Drosophila. J. Hered. 62: 41-44. gested by previous work (XUet al. 1990) and indicates HELD,L. I., JR., and P. J. BRYANT,1984 Cell interactions con- trolling the formation of bristle patterns in Drosophila, pp. that deltex is likely to play an important role in the 291-322 in Pattern Formation, edited by G. M. MALICINSKIand same genetic circuitry within which these three neu- S. V. BRYANT.Macmillan, New York. rogenic loci operate. JAN,L. Y., and Y.N. JAN, 1982 Antibodies to horseradish per- oxidase as specific neuronal markers in Drosophila and in gras- We would like to thank ABRAHAMSCHALET for kindly providing shopper embryos. Proc. Natl. Acad. Sci. USA 72: 2700-2704. US the dgMallele; the GOODMANlaboratory for antibody reagents JOHANSEN, K., R. G. FEHON and S. ARTAVANIS-TSAKONAS, and T.N. SCOTTGALE,C. DELIDAKIS,I. DAWSON and RUTHSCHLES- 1989 The Notch gene product is a glycoprotein expressed on INGER-BRYANT their for helpful comments on the manuscript. We the cell surface of both epidermal and neuronal precursor cells are grateful to all our colleagues in the ARTAVANISlaboratory as during Drosophila development. J. Cell Biol. 109: 2427-2440. well as the KANKEL laboratory for valuable suggestions, to W. J~GENS,G., E. WIESCHAUS,C. NUSSLEIN-VOLHARDand H. KLUDIG, BARRYPIEKOS for scanning electron microscopy. We would also 1984 Mutations affecting the pattern of larval cuticle in Dro- like to acknowledge the expert technical assistance of RUTHSCHLES- sophila melanogaster. 11. Zygotic loci on the third chromosome. INGER-BRYANT,WAN YU andJIAXIN CAI.T.X. was supported by a Wilhelm Roux's Arch. Dev. Biol. 193: 283-295. Yale University doctoral fellowship. This research was partly sup- KELLEY,M. R., S. KIDD, W. A. DEUTSCHand M.W. YOUNG, ported by National Institutes of Health grants GM29093 and NS 1987 Mutations altering the structure of epidermal growth 26084. factor-like coding sequences at the Drosophila Notch locus. Cell 51: 539-548. KEPPY, D. O., and W. J. WELSHONS,1977 The cytogenetics of a LITERATURE CITED recessive visible mutant associated with a deficiency adjacent to theNotch locus of Drosophila melanogaster. Mol. Gen. Genet. ARTAVANIS-TSAKONAS,S., 1988 The molecular biology ofthe 181: 319-324. Notch locus and the fine tuning of differentiation in Drosophila. KIDD, S., M. R. KELLEYand M. W. YOUNG,1986 Sequence of the Trends Genet. 4 95-100. Notch locus of Drosophila melanogaster: relationship of the en- BREEDEN,L., and K. NASMYTH,1987 Similarity between cell cycle coded protein to mammalian clotting and growth factors. Mol. genes of budding yeast and fission yeast and the Notch gene of Cell. Biol. 6: 3094-3 108. Drosophila. Nature 329 651-654. KIDD, S., and M. W. 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