notch locus of drosophila is required in epidermal cells ...876 p. e. hoppe and r. j. greenspan...

Development 109, 875-885 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

875

The Notch locus of Drosophila is required In epidermal cells for epidermal

development

PAMELA E. HOPPE1* and RALPH J. GREENSPAN2§1 Department of Biology, Princeton University, Princeton, NJ 08544, USA2Department of Neurosciences, Roche Institute of Molecular Biology, Nutley, NJ 07110, USA

•Current address: Department of Genetics, Box 8232, Washington University School of Medicine, 660 S. Euclid Avenue, St. Louis, MO63110, USA§ To whom all correspondence should be addressed

Summary

The Notch locus of Drosophila plays an important role incell fate decisions within the neurogenic ectoderm, a rolethought to involve interactions at the cell surface. Wehave assayed the requirement for Notch gene expressionin epidermal cells by two kinds of genetic mosaics. First,with gynandromorphs, we removed the wild-type genelong before the critical developmental events to producelarge mutant clones. The genotype of cells in large cloneswas scored by means of an antibody to the Notchprotein. Second, using mitotic recombination, weremoved the gene at successively later times aftercompletion of the mitotically active early cleavagestages, to produce small clones. These clones weredetected by means of a linked mutation of cuticle

pattern, armadillo. The results of both experimentsdemonstrate a requirement for Notch expression byepidermal cells, and thus argue against the model thatthe Notch product acts as a signal required only in theneuroblast to influence neighboring epidermal cells. Themitotic recombination experiment revealed that Notchproduct is required by epidermal cells subsequent toneuroblast delamination. This result implies that theNotch gene functions to maintain the determined state ofepidermal cells, possibly by mediating cell surface inter-actions within the epidermis.

Key words: neurogenesis, mosaics, determination,Drosophila, Notch, epidermal development.

Introduction

The Notch locus of Drosophila is required for theproper assignment of developmental fates in ectoder-mal cells. During embryogenesis its activity is neededfor the correct parcelling of cells within the neurogenicectoderm into neural and epidermal fates. In normaldevelopment, the neuroblasts, the precursor stem cellsof the embryonic nervous system, arise in a stereotypedpattern from the neurogenic ectoderm located on eitherside of the ventral midline (Poulson, 1950; Thomas et al.1988; Hartenstein and Campos-Ortega, 1984; Doe etal.1988). The neuroblasts move interiorly and divide,producing the neurons of the brain and ventral nervecord. The cells within the neurogenic region thatremain at the surface of the embryo become dermato-blasts and give rise to the epidermal layer, whichsecretes the embryonic cuticle.

Embryos homozygous for a deletion of the Notchlocus develop a greatly hypertrophied nervous systemas a result of the misrouting of epidermal precursorsinto the neural pathway of development (Poulson,

1940). Similar phenotypes are produced by six otherzygotically acting loci (Lehmann etal. 1981; Lehmann etal. 1983). Because deletion of any of the neurogenic lociproduces the same neuralized phenotype, and becausegenetic interactions occur between mutations in differ-ent neurogenic loci (Vassin et al. 1985; de la Concha etal. 1988), the products of these loci are believed to becomponents of a common process that guides cellswithin the neurogenic ectoderm into the proper devel-opmental pathway.

Cell-cell interactions appear to play a central role inthe determination of these developmental fates. Ab-lation studies in the grasshopper have shown that, whena neuroblast is killed, a neighboring undifferentiatedectodermal cell changes its fate to assume that of theabsent neuroblast (Doe and Goodman, 1985). Thisresult implies that the neuroblast somehow communi-cates its presence to surrounding cells and therebyprevents them from becoming neuroblasts. The neuro-genic loci of Drosophila may encode componentsrequired for the interaction between neuroblast andsurrounding cells. Mutations in these genes would thus

876 P. E. Hoppe and R. J. Greenspan

impair cell communication, allowing epidermal cells toassume the neuroblast fate.

The molecular characterization of the locus providesfurther evidence that Notch plays a role in cell surfaceinteractions. The sequence of the Notch transcriptpredicted (Wharton et al. 1985; Kidd et al. 1986), andsubsequent biochemical studies have identified (Kidd etal. 1989; Johansen et al. 1989), a very large transmem-brane protein (2703 amino acids).

Because Notch is believed to mediate an interactionbetween developing cells, it is crucial to define accu-rately the time and place of its action. To elucidate therole of the Notch product, we wanted to perform afunctional test to determine which cells in the neuro-genic region required the Notch protein for correctneuroblast segregation. One model is that the Notchproduct may be required in the neuroblast to signaladjacent cells to become epidermis, but that it is notrequired in the epidermal cells themselves for properdifferentiation. This proposal is supported by cell trans-plantation experiments in which single cells mutant forNotch were able to form epidermis when transplantedinto wild-type hosts (Technau and Campos-Ortega,1987).

However, the alternative view, that expression ofNotch is required autonomously by epidermal cells tofollow the epidermal pathway, was suggested by theresults of a gynandromorph mosaic experiment (Hoppeand Greenspan, 1986). In that experiment, a cuticlemarker was used to assess the ability of Notch~ cells todifferentiate as epidermis in gynandromorph mosaicembryos. No Notch~ cells produced cuticle within theneurogenic region, indicating that the gene's functionwas required autonomously by epidermal cells in largeclones to complete differentiation.

By relying on a cuticle marker scorable only at theend of embryogenesis, it is conceivable that we couldhave missed local nonautonomy at clonal borders be-cause erosion at the edge of the cuticle would havedestroyed the evidence for mutant epidermal cells. Toaddress this possibility, we have now investigated cellfate decisions along borders of large clones at a muchearlier stage when we can score epidermal cells directly.Specifically, we have repeated the gynandromorphmosaic experiment, but have used an antibody specificfor the Notch protein (Johansen et al. 1989) to dis-tinguish wild-type and mutant cells shortly after neuralsegregation has occurred. We could thereby assess theability of Notch' cells to initiate epidermal develop-ment by examining cells at mosaic borders early inembryogenesis to determine whether mutant cells hadentered the epidermal pathway.

To investigate whether epidermal cells require theNotch product after the events of neural segregationhave occurred, we have made use of mitotic recombi-nation as an alternate, finer tool to produce mosaics. Inthis experiment, we induced small clones of Notch~cells by irradiating embryos heterozygous for a nullallelle of the locus.

Materials and methods

Induction of clonesVirgins of the genotypes w armYD3s NXK"/FM7 and warmVD351FM7 were mated to Oregon R males for 2 days,moved to collecting chambers (see Wieschaus and Nusslein-Volhard, 1986) and placed at 22°C for at least 1 day. Afterseveral changes of fresh plates, eggs were collected for 30 minperiods, then allowed to develop at 22°C for 3, 4, or 5 h fromthe midpoint of the collection. The embryos were irradiatedfor 4 min at approximately 350 radmin"1, placed at 25°C forapproximately 12 h, washed, dechorionated with bleach, andplaced in water to complete development (see Gergen andWieschaus, 1985). Because the frequency of clone inductiondepends upon the number of cells that are in the appropriatephase of the cell cycle at the time of irradiation, and the size ofthe clone at the end of development depends upon thenumber of cell divisions that follow, the survival of thearmadillo* Notch~ clones is gauged by comparison to marker{armadillo' alone) clones induced at the same timepoint ofirradiation. To control for possible occurrence of false clones,eggs laid by Oregon R flies were collected and irradiated in anidentical fashion. In addition, several collections from w armN/FM7 females were processed without being irradiated. Todetermine the developmental stage at each timepoint ofirradiation, a few collections were fixed by the methanol mass-devitellinization procedure (Wieschaus and Nusslein-Vol-hard, 1986), stained with 0.1% toluidine blue O in 20%ethanol for 30 s, dehydrated in ethanol, and mounted in 1:1Canada balsam: methyl salicylate for examination.

Cuticle preparations were made according to Van der Meer(1977). Experimental and control progeny from each timepoint of irradiation were mounted separately on slides. Toprevent any bias in the scoring of clones, the labels on allslides were covered and the slides scrambled prior to examin-ation. The number of clones in each animal and the number ofdenticles in each clone were recorded.

The comparison of Notch~ and control clones via cumulat-ive distribution curves (Fig. 8) required that control values beadjusted to reflect clones expected in a given number ofcontrol animals, since the number of control animals scoredwas never exactly equivalent to the number of experimentals.Thus, the 3h values were multiplied by 245/217 to estimatethe number expected in 245 animals rather than the 217actually scored. Similarly, the 4h controls were multiplied by146/301 and 5 h controls by 171/205.

Antibody staining and sectioning of gynandromorphembryosA rabbit antiserum raised against a fusion protein containing aportion of the Notch coding sequence from the EGF-repeatregion was obtained from Kristin Johansen in the laboratoryof Spyros Artavanis-Tsakonas. The staining protocol used wasthe same as reported by Johansen et al. (1989) with theexception that antibody was used at a higher concentration(1:50 in PBS containing 1 % BSA), the Vectastain Elite ABCkit was used to amplify the signal, and development timeswere longer, up to 1 h. To eliminate nonspecific staining thatcould appear under these conditions, the antiserum waspreabsorbed with mass-devitellenized embryos of 16 h ormore of age collected from the NXK"/FM7 stock. Theseadditional steps were necessary to produce No/c/i-specificstain of sufficient intensity to detect in plastic-sectionedembryos. Specificity of the antibody stain in our hands wasconfirmed in two ways. First, no staining appeared wheneither primary or secondary antibody was omitted from the

Epidermal requirement for Notch 877

procedure. Second, the mosaic-generating cross (describedbelow) produces a significant number of completely Notch'animals, recognizable by their neuralized phenotype, thatexhibit no staining in any tissue. In addition, each mosaicanimal is in itself a control for Notch specificity becauseneuralized mutant regions contain nonstaining cells such asdorsal epidermis or mesoderm while wild-type regions of thesame animal exhibit staining in these same tissues.

Virgins of the genotype y NXK"/FM7 were mated to malescarrying the unstable ring-X chromosome, In(l) wvC/N+Y,and placed in collecting chambers (see above) at 25°C. Eggswere collected for 2h periods, then aged for 7h from theendpoint of the collection. Collections were washed, dechor-ionated in bleach, and fixed in 2 ml of Bouin's fixative (Sigma)and 4 ml heptane for 15min, transferred to double-stick tapeand devitellinized under fresh Bouin's with a sharpenedtungsten wire. After removing fixative with washes of PBS,blocking serum (Vector Vectastain ABC Kit) was applied forat least lh. The embryos were incubated with preabsorbedprimary antiserum for 5h, rinsed lh (PBS with 1% BSA),incubated with biotinylated secondary antibody (VectastainKit) for 2h, rinsed for 30min, placed in ABC reagent(Vectastain Elite Kit) for 45 min, rinsed in 3 changes of PBSwith 1 % BSA for a total of 20 min, then placed in CATbuffer(50 mM ammonium acetate brought to pH5.5 with citric acid,0.1% Tween 20). The peroxidase stain was developed in0.06% hydrogen peroxide, 0.05% diaminobenzidine (Sigma)in CATbuffer for up to 1 h. Embryos were embedded in Epon812 and cut in 2 or 3 micron sections as described byWieschaus and Sweeton (1988). The sections were counter-stained with toluidine blue-borax for 10 min on a warm plate,then destained for 15 min (see Altman and Bell, 1973) and air-dried prior to applying DPX mountant (Gurr) and a coverslip.

Results

Epidermal cells along gynandromorph borders alwaysexpress NotchIf Notch expression is required only by neuroblasts as asignalling protein, then mutant epidermal cells shouldbe present at gynandromorph borders (Fig. 1). Toascertain the ability of mutant cells along the border ofa large Notch~ clone to enter the epidermal pathway,we used an unstable ring-A' chromosome to producemosaics containing large areas of mutant cells. Thegenotype of the cells was assessed by staining themosaics with an antibody specific for the Notch protein(Johansen et al. 1989; also see Methods). Since theNotch product is a transmembrane protein bound to thecell, the antibody serves as a cell autonomous marker.Neuroblasts and epidermal cells were distinguished bymorphological criteria in plastic sections. As neuro-blasts delaminate, they enlarge, become round andbegin dividing, producing smaller daughters dorsally. Incontrast, epidermal cells are small, divide within theplane of the embryonic surface and initially adopt amore rectangular shape within the epithelium beforebecoming quite flat, spreading over the embryo's sur-face (Poulson, 1950; Campos-Ortega and Hartenstein,1985).

The animals were permitted to develop for at least7 h, at which time the process of neuroblast segregationis virtually complete (Hartenstein and Campos-Ortega,

1984). Large numbers of them were fixed, stained andembedded, and after being viewed under a dissectingmicroscope, seven embryos containing a considerablelength of mosaic border within the neurogenic regionwere selected for cross-sectioning. The sections wereexamined, and at each point where the border ofantibody staining fell within the neurogenic region ofthe embryonic thorax or abdomen, we scored themorphology of cells on either side as neural or epider-mal.

All of the epidermal cells along these borders wereNotch+. If Notch~ cells had been able to initiateepidermal development, we would have expected tofind small, unstained cells that had remained at theembryonic surface and joined the wild-type epidermallayer. However, despite the examination of more than200 border points sampling over 100 border epidermalcells, not one Notch~ epidermal cell was found (Fig. 2).

To maximize the possibility of detecting rare, mutantepidermal cells, we limited our analysis to those bordersmost likely to harbor such a cell. This excluded the largenumber of mosaic borders falling at the ventral midline,due to the prior invagination of mesodermal andmesectodermal cells at this point. Because cell-cellinteractions may not occur between cells on either sideof the midline, a mosaic border at the ventral midlinemay not test the autonomy of Notch gene function. Inaddition, we excluded borders that fell in the mostdorsal area of the neurogenic region, due to the

A. Notch is required only In the neuroblast

V. mutant cells remain Inepidermis when In contactwith a wt nauroblast

B. Notch is required autonomously by epidermal cells

ALL mutant c*ta becomeneurobtasts

Fig. 1. The configuration of mutant and wild-type (wt) cellsat mosaic borders tests the autonomy of Notch action in theepidermis. (A) If the Notch protein is required only by theneuroblast to signal adjacent prospective epidermal cells,some mosaic borders will appose a wild-type neuroblast andmutant prospective epidermal cells, resulting in Notch~ cellsdifferentiating as epidermis at the mosaic border. (B) Ifepidermal cells in the neurogenic region require Notchproduct autonomously, then all mutant cells at all mosaicborders will become neuroblasts. Small ellipses representprospective epidermal cells, large circles representneuroblasts and shading indicates mutant genotype.


difficulty in precisely locating its dorsal limit. Outside ofthis limit, epidermal differentiation is not dependent onthe action of the Notch locus (Lehmann et al. 1981;Hoppe and Greenspan, 1986). Our failure to find anymutant epidermal cells in the neurogenic region indi-cates that in large clones each epidermal cell, eventhose along the mosaic border, must express the Notchprotein.

Among the more than 200 borders scored, it was notpossible to determine directly at any given border pointwhether a Notch+ neuroblast was ever present where itmight have permitted a Notch~ epidermal cell tosurvive. We were unable to score neuroblast genotypedirectly because, although all cells in the neurogenicregion initially stain with antibody during neuroblastsegregation (Kidd etal. 1989; Johansen etal. 1989; andour unpublished observations), at the time neuroblastsegregation is complete even wild-type neuroblasts failto stain (Fig. 3). (The lack of staining in neuroblasts atthis later time could probably not be discerned in wholemount preparations because of surface epidermal stain-ing. Antibody staining of neurons is evident in laterstages, particularly along their axons; Kidd et al. 1989;Johansen et al. 1989; and our unpublished obser-vations.) However, given the separation of neural andepidermal lineages, as well as the randomness of mosaicborders, if it were possible for a mutant cell to differen-tiate as epidermis under the influence of an adjacentwild-type neuroblast, it is virtually certain that wewould have seen it among the more than 100 epidermalcells sampled. The failure to find separation betweenNotch+ genotype and epidermal phenotype in over 100cells means that if the genotype of some other cell wascritical, that cell must map <1 sturt from the epidermalcell. Adjacent cells in the blastoderm map at least 2-3sturts apart (Szabad et al. 1979), so it is unlikely that thegenotype of some other cell (e.g. a neuroblast) isimportant. [The sturt is a measure of developmentalrelatedness based on the probability of a mosaic borderpassing between any two points on the blastoderm. It isconceptually related to a genetic map unit, and issimilarly calculated (Garcia-Bellido and Merriam,1969).]

This analysis of mosaic borders in gynandromorphsshortly after neural segregation confirmed the auton-omy of Notch in epidermis observed in cuticle prep-arations (Hoppe and Greenspan, 1986). The previousmosaic study, however, had indicated that in addition tothe lack of mutant cuticle, a loss of wild-type cuticleoccurred prior to the end of embryogenesis. Theclearest indication of this loss was the lack of isolated'islands' or 'peninsulas' of wild-type cuticle in mosaicanimals. It was unknown whether the deficit of wild-type cuticle was the result of the misrouting of wild-typecells at the time of neural segregation, of a later failureof differentiation, similar to that described for dorsalepidermal cells in neurogenic mutants (Jimenez andCampos-Ortega, 1982), or of actual erosion of cuticle.

Examination of the mosaic borders in antibody-stained sections allowed us to detect certain configur-ations of wild-type epidermis that were not observed in

cuticle mosaics, those that contained an isolated 'island'of wild-type cuticle surrounded by mutant cells. Anumber of the sections contained small ventral patchesof wild-type epidermal cells not connected to the dorsalregion of epidermis unaffected in Notch~ embryos(Fig. 4). The observation that isolated patches of wild-type cells can initiate epidermal development suggeststhat our previous report of a loss of wild-type cuticle inmosaics (Hoppe and Greenspan, 1986) is a later,secondary degenerative event, rather than the result ofthe misrouting of wild-type cells during neural segre-gation. Moreover, the presence of epidermal islands inthe preparations argues that our failure to detectNotch~ epidermal cells at borders is not due to loss ofthese cells following segregation.

Notch product is required in epidermal cells afterneuroblast delaminationTo investigate further the role of the Notch gene in theepidermis, we induced clones consisting exclusively ofepidermal cells later in development by mitotic recom-bination (Fig. 5). Therefore any effect of the Notchmutation in these clones, none of which are associatedwith a mutant neuroblast, cannot be the result of theNotch product functioning solely as a signal fromneuroblasts to epidermal cells. For detection of mitoticclones in the epidermis, the Notch mutation was linkedto a null allele oiarmadillo, a cell autonomous 'segmentpolarity' gene (Nusslein-Volhard and Wieschaus, 1980;Wieschaus et al. 1984; Wieschaus and Riggleman,1987). We determined that embryos mutant for bothgenes exhibited the neuralized phenotype characteristicof Notch~ embryos, as to be expected since there is noepidermis to form denticles. Because of their closelinkage, any induced Notch~ clones would also behomozygous for the armadillo mutation. Such cloneswould produce clusters of ectopic denticles in regions ofnaked cuticle, but only if genotypically Notch~ cells cansurvive and secrete cuticle.

Synchronously aged collections of embryos wereirradiated at several different timepoints. Irradiationsperformed during cleavage stages resulted in aborteddevelopment in the majority of embryos. The firstsuccessful irradiations were at the 3h timepoint, whenvirtually all embryos (61/64) were in the process ofcellularization, embryonic stage 5 (Campos-Ortega andHartenstein, 1985; Wieschaus and Nusslein-Volhard,1986; and see Fig. 6), as ascertained by examination offixed collections (see Methods). At this age all blasto-derm nuclei are in interphase of mitotic cycle 14 (Foe,1989; and see Fig. 6). Therefore, recombination eventsinduced at this time result in the production of onemutant daughter, the first homozygous Notch~ cell,upon completion of the 14th mitosis. Since the majorityof neuroblasts delaminate prior to the completion ofcycle 14 (Hartenstein and Campos-Ortega, 1984), thetuning of this division and the fact that only onedaughter cell can be mutant means that clones will berestricted to either the neuroblast or the epidermallineage. Fig. 5 illustrates the possible configurationsthat produce this result. Because the armadillo marker

ms

WT

vm

Fig. 2. Examination of mosaic borders revealedno unstained (mutant) cells in the epidermallayer. In A, a mutant patch lies on one side ofthe ventral midline (vm). The wild-typeepidermal cell at the mosaic border is indicatedby the small arrow. To the right, this cell isadjacent to wildtype epidermal cells that form asmooth epithelium of small cells with smallnuclei. To the left of this border cell, theexternal outline of the embryo becomesirregular, and the unstained cells, indicated byarrowheads, are large cells with large nucleithat have the rounded appearance ofneuroblasts. No unstained cells have joined theepidermal layer at its border. In B, a nearbysection is shown where, again, the wild-typeepidermal cell at the mosaic border is indicatedby the small arrow. The unstained cells at themosaic border, indicated by arrowheads, havenot joined the smooth epidermal epithelium,and like neuroblasts are dividing to producedaughters dorsally (interiorly).Fig. 3. Neuroblasts do not stain with the Notchantibody after neural segregation is complete.A section of a Notch mosaic where involutionat the ventral furrow during gastrulation causedthe gynandromorph border to fall at the ventralmidline (vm) of the ectodermal anlagen isshown. All ectodermal cells on the right (N~)side fail to stain with the Notch antibody,although mesodermal (ms) stainingdemonstrates sufficient permeability to theantibody. On the wild-type (WT) side, theepidermal layer of cells continues to stain withthe antibody (small arrows) but the neuroblasts(arrowheads) do not. The staining detectsNotch protein in the cytoplasm as well as on thesurface of epidermal cells, which containsignificant Notch transcript at this time (Hartleyel al. 1987); this is probably due to themeasures taken to intensify the antibody signal(see Methods).

Fig. 4. A small island of wild-type cells is ableto initiate epidermal development. In thesection shown, involution at the ventral furrowduring gastrulation has produced a small groupof wild-type ectodermal cells at the ventralmidline, and a few cells (small arrows) haveinitiated epidermal development. Mesodermalstaining (ms) is also evident.

A. 00000NB

SEG DIV

NB

B.DIV


NB

c. 00000 —DIV

SECONDNB

SEG ^

Fig. 5. Irradiation in cycle 14 can produce mutant clones that contain epidermal cells or neural cells, but not both. In eachcase illustrated, irradiation has produced a chromosomal exchange event (asterisk) in a blastoderm cell (rectangular shape).Only one genetically mutant cell is produced upon division of the target cell (mutant cell indicated by shading). In A and B,cell division (DIV) occurs after segregation of the majority of the neuroblast population (NB SEG). In A, the mutant cell isa prospective epidermal cell (indicated by ellipses); any mutant progeny that survive in the epidermis will be detected by thearmadillo marker. In B, the mutant cell is produced upon the first division of the NB, generating either a mutant NB or amutant ganglion mother cell. Neither of the events in B are detected by the armadillo marker; thus our experiment does nottest the effect of Notch in strictly neural clones. In C, the target cell completes cycle 14 on the blastoderm surface before theend of neuroblast segregation. The one mutant daughter will either become a NB, an event not detected by the armadillomarker, or initiate epidermal development, in which case any mutant progeny that differentiate in the epidermis will bedetected by the armadillo marker. In no case is a marked (mutant) epidermal cell adjacent to a mutant NB. Therefore, ourexperiment tests the effect of the Notch mutation on the development of strictly epidermal clones.

detects mutant epidermal cells only, recombinationevents in the neural lineage do not produce detectableclones and could not be studied (Fig. 5B). Therefore,our study is restricted to small epidermal Notch~ clonesthat develop in the context of a fully wild-type comp-lement of neuroblasts (Fig. 5A,C). Even in the casewhere a neuroblast delaminates only after completingcycle 14 on the embryonic surface (Technau and Cam-pos-Ortega, 1986), the mutant clone produced willcontain exclusively neural or epidermal cells (Fig. 5C).Any effect of the Notch mutation on the number andsize of these clones would have to reflect a requirementfor Notch product in the epidermis.

The timing of mitoses in this cell population alsomeans that virtually all neuroblasts will have delami-nated by the time clones become homozygous. Theywill have left the blastoderm layer in the SI and S2waves (see Fig. 6 and also Hartenstein and Campos-Ortega, 1984 and Foe, 1989), which precede the earliestpossible homozygosing of clones. Thus, any effect ofthe Notch mutation on induced clones would alsoreflect an epidermal role for the gene product sub-sequent to any interactions with presumptive neuro-blasts.

Irradiation at the 3 h timepoint resulted in a very highfrequency of armadillo' clones in control animals (0.48clones/total progeny), one or more clones being pres-ent in virtually every heterozygous animal (Table 1).Some clones were also identified in experimental ani-mals, indicating that a few genotypically armadillo'

Notch cells were able to complete epidermal develop-ment and secrete cuticle (Fig. 7).

However, comparison of the experimental and con-trol clones revealed that the number of Notch~ clonesscored was sharply lower (frequency=0.21), indicatingthat just over half of the expected clones failed todifferentiate in the epidermis. In addition, the Notch~clones that did appear were smaller, judging from theaverage number of denticles produced (4.5 denticles/clone), compared to controls (6.4 denticles/clone).Denticle morphology and density were similar in exper-imental and control clones. The predominance of smallclones in the Notch~ sample is evident in a comparisonof the cumulative distribution curves of experimentaland control clones (Fig. 8A). Since each epidermal cellis thought to secrete about 3 denticles (Wieschaus andRiggleman, 1987), most of the surviving Notch' clones(80%) are one or two cells in size.

The 4h timepoint contained embryos in early gastru-lation, stages 6, 7 and 8. At this time the cells of theneurogenic region are still in cycle 14 (Foe, 1989). Incontrols, the irradiation at this time produced substan-tially fewer clones than controls at 3h, but they werestill the same size (Table 1, Fig. 8B). This suggests that,although no cell division has occurred, the number ofcells that are targets of mitotic recombination is lower at4h than at 3h, but that those remaining responsive willundergo the same amount of proliferation. Notch~clones, in contrast, appeared at a frequency equal tocontrols (0.14 and 0.16, respectively), but were signifi-


Irradiations 3 hr 4 hr 5hr

clonesmissing

and small

clonessmall

ectodermappears

'neurogenlc'

S1nb's

delam

hrat22°C0 1

earliest possiblehomozygous clones

clonesnormal

mitoses

domalnmitoses

S2 nb'sdelam. ,

S3 nb'sdelam.

10

Stages10

%X\\ ^ \ V

cephalic furrow 9> o^appears

\

\Fig. 6. Time course of embryogenesis with reference to Notch clone induction, neuroblast delamination and mitosis in theneurogenic region. SI, S2 and S3 refer to waves of neuroblast delamination, SI constituting 61 % and S2 constituting 21 % ofthe total (Campos-Ortega and Hartenstein, 1985). Thus, 82% of the neuroblasts have delaminated by the time the firstmitoses begin in the neurogenic region ('N' and 'M' domains of Foe, 1989).

Table 1. Notch and control clones marked with armadilloTimepoint ofirradiation

Number ofclones

Clonefrequency

Clonesize S.E.

3 h cycle 14blastoderm

4 h cycle 14early gastrula

5 h cycle 14-15stomodeal invag.

controlNotch'

controlNotch'

controlNotch'

9350

4721

4845

0.430.21

0.160.14

0.230.26

6.394.47

6.174.38

5.635.16

(0.42)(0.48)

(0.58)(0.64)

(0.54)(0.48)

Clone frequency represents the number of clones per total animals scored. Clone size is expressed in average number of denticles perclone. s.E. denotes standard error of the mean.

* Notch' clones significantly smaller than controls, Mest P=0.0026.** Notch' clones significantly smaller than controls, f-test /)=0.025.

cantly smaller on average (4.4 vs. 6.2 denticles/clone)(Table 1, Fig. 8B). This indicates that, although theexpected number of Notch~ clones survive at this timepoint, the cells completed fewer divisions or somemembers of the clone were lost.

The 5h collection spanned the time of stomodealinvagination, containing stage 9 and 10 embryos. Ir-radiation at this time could result in recombination incells still in cycle 14 or in cells that have divided and

entered cycle 15. Notch and control clones induced atthis stage were indistinguishable in frequency and size(Fig. 8C). The similarity in behavior of Notch~ cellsand control cells suggests that sufficient zygotic ex-pression of the gene has occurred by the time theydivide. The fact that irradiations at these three differenttimes give different results, even though they all pre-cede cell divisions (see Fig. 6), conforms with theobserved asynchrony of mitosis in this region during

** A

* \

\ » ' " , , ' . , » . <

»>

< -

Fig. 7. /Vofc/i and control clones induced at 3 h andmarked with armadillo result in the appearance of ectopicdenticles in regions of naked cuticle. In A, the arrowindicates a Notch' clone comprising 5 denticles. In B, thearrow indicates a control clone comprising 15 denticles.

cycle 14 (Foe, 1989) and with differential susceptibilityof cells to mitotic recombination due to their place inthe cell cycle.

Discussion

The product of the Notch locus takes part in thespecification of embryonic cell fate through its ex-pression by presumptive epidermal cells. The timecourse of its requirement by these cells argues that it isinvolved in the maintenance of the determined epider-mal state. These two conclusions follow from analysis ofmosaic experiments presented here. In conjunctionwith what is known about the structure of the geneproduct, these results suggest that the Notch proteinmay act by stabilizing contacts between cells in theepithelial sheet of epidermal precursors.

Notch expression is required in epidermal precursorsTwo kinds of mosaic analysis have been carried out to

en

a>nE

a>>

Eu

100 -

80-

60-

40-

20"

0


A 3hr

60-

40-

20-

0

B 4hr

arm N

6 0 -

4 0 -

2 0 -

0 -

c

, r=J-i

5 h r

arm N

M~^ arm

10 15 20

Size of Clone

(number of denticles)

Fig. 8. Cumulative distributions of clones. In each graph,cumulative distribution curves are used to compare the sizedistribution of Notch~ and control clones obtained at agiven timepoint. Because the number of clones dependsupon the number of animals scored, the comparisons weremade by drawing the true distribution of Notch~ clones andadjusting the control values to reflect the clones expected ina control population exactly the same size as theexperimental population (see Methods). (A) Irradiation at3 h produces fewer Notch~ clones in virtually all sizeclasses, as shown by the difference in the height of the twocurves. Those Notch~ clones that are obtained are alsosmaller, the curve reaching nearly maximum height earlier,in the small clone sizes. (B) Irradiation at 4h producesequal numbers of clones, but, as reflected in the faster riseof the Notch' curve, the Notch~ clones are smaller (seealso Table 1). (C) Irradiation at 5h produces Notch' clonesthat are indistinguishable from controls.

determine which cells require Notch expression forcorrect specification of cell fate. When assayed with anantibody to the Notch protein, large clones in gynan-dromorphs show a strict correlation between Notch+

genotype and proper epidermal development. To scoreaccurately as many border cells as possible, we re-


covered sections and reconstructed the relevant cloneboundaries from seven mosaics. If the genotype ofsome other cell were crucial, we would have detectedsome Notch~ epidermal cells at these borders.

In mitotic recombination experiments, over half ofthe expected epidermal clones induced by early ir-radiation fail to appear, and those that are recoveredare smaller than controls. The timing of cell division inthe neurogenic region is such that these mitotic clonesare restricted to the epidermal lineage (Fig. 5). Thereduction in epidermal clone number and size cannot,therefore, be attributed to the genotype of any othercell type.

Both experiments concur that wild-type gene productmust be expressed by the cells that will become epider-mis. They are consistent with recent mosaic findingsconcerning the neural-epidermal decision in imaginaldiscs, demonstrating a requirement for Notch productin cells choosing the epidermal fate (P. Heitzler and P.Simpson, personal communication). Moreover, all ofthese experiments contradict a model ascribing the siteof Notch gene action exclusively to neuroblasts (Tech-nau and Campos-Ortega, 1987; Campos-Ortega, 1988).

Our results are also in good agreement with compar-able mosaic experiments done in C. elegans with twogenes homologous to Notch. The genes lin-12 and glp-1are known to be required for cell fate decisions in whichcell interactions play an important role (Kimble, 1981;Greenwald et al. 1983; Austin and Kimble, 1987; Preissetal. 1987; Seydoux and Greenwald, 1989). Both lin-12and glp-1 are transmembrane proteins that containportions of both the intracellular and extracellulardomains that are homologous to Notch (Greenwald,1985; Yochem et al. 1988; Yochem and Greenwald,1989; Austin and Kimble, 1989).

In mosaics, the lin-12 locus has been shown to berequired autonomously in the VU cell for VU develop-ment (Seydoux and Greenwald, 1989), while glp-1 wasfound to act in the germ line for regulation of the germline by the distal tip cell (Austin and Kimble, 1987).Thus, both genes must function in the cell 'responding'to the signal, though it is not known if they are the'receptors'. Notch, by analogy, acts in the 'responding'epidermal precursor. One interesting observation dis-cussed by Seydoux and Greenwald (1989) was that inmosaic worms containing one mutant and one wild-typecell in the interacting pair, the mutant cell alwaysbecame the anchor cell. One possibility they raised wasthat initially there is symmetrical expression of lin-12 inboth cells, which eventually tilts towards a preponder-ance of expression by the VU cell and a reciprocalreduction in the anchor cell. This idea has receivedsome recent support in Drosophila from mosaic exper-iments in imaginal discs employing duplications of theNotch locus (P. Heitzler and P. Simpson, personalcommunication). In these experiments, cells containingmore copies of wild-type Notch preferentially adoptedthe epidermal fate. Because neuroblasts originally stainfor Notch, but lose staining at least transiently aftersegregation (Fig. 3), one can speculate that the disap-pearance of Notch activity in neuroblasts may be as

important as its persistence in epidermal cells to specifycorrect cell fate.

Notch is required beyond the time of neuroblastdelaminationThe fact that our mitotic clones do not lose their wild-type Notch allele until after neuroblast delamination isvirtually complete (Fig. 6) provides us with new infor-mation on the time course of the gene's action. It arguesthat there is an ongoing requirement for Notch geneproduct in epidermal cells for a period of time followingthe initial interactions between equivalent cells in theneurogenic ectoderm. This can best be thought of as arole in maintaining the determined state.

There is already good reason to believe that theneural-epidermal decision requires stabilization.Heterochronic transplants of ectodermal cells betweenneurogenic and non-neurogenic regions demonstratethat even after neuroblast delamination, a presumptiveepidermal cell can change fate and become a neuroblast(Technau et al. 1988). Similarly, temperature-shift ex-periments with the mutant shibire" have defined a long-term requirement for this gene's activity in normalneurogenesis, extending past the end of neuroblastsegregation (Poodry, 1990). Our experiments supportthe idea that Notch mediates such a stabilization, sincethe removal of Notch* activity in presumptive epider-mal cells, even after the vast majority of neuroblastshave delaminated (Fig. 6), results in loss of these cellsfrom the epidermis. Moreover, Notch is known to act atmultiple times in development. Studies of a tempera-ture-sensitive allele and of mitotic clones induced inimaginal disks have demonstrated a requirement inlater stages of embryonic (Shellenbarger and Mohler,1978), and larval development (Portin, 1980; Dietrichand Campos-Ortega, 1984; Cagan and Ready, 1989).Analysis of the appearance of Notch transcript andprotein has also revealed that it is expressed at laterstages of development and continues to be present inepidermal cells after the completion of central neuro-genesis (Fig. 3; Hartley et al. 1987; Kidd et al. 1989;Johansen et al. 1989).

Whether Notch is also involved in the initiation aswell as the maintenance of the determined state inepidermal cells remains an open question. None of themosaic experiments have been able to address thisissue. In the case of lin-12, mosaic experiments didprovide evidence for a role in initiation of the deter-mined state of the VU cell. In all of the mosaicsexamined, Seydoux and Greenwald (1989) found thatwhenever one cell of the pair was mutant, it alwaysbecame the anchor cell and the other always became aVU cell. If the gene were required only for mainten-ance, and initiation occurred independently, then itshould have been possible to obtain mosaics in whichonly one cell was mutant but both were anchor cells.The available evidence for Notch and lin-12 is compat-ible with both genes playing roles in initiation andmaintenance of the determined state.


Survival of some genotypically Notch clones is due toperduranceA question remains as to why some of our Notch~clones do survive after irradiation at the earliest timepoint. The most likely explanation is perdurance ofNotch+ product in genotypically mutant cells. Becauselarge clones probably derive from mutant precursorcells that were both established early, prior to signifi-cant zygotic Notch expression, and subject to a greaternumber of subsequent cell divisions, which furtherdilute the product, cells of a large clone would be lesslikely to contain sufficient Notch product to completeepidermal development.

This explanation conforms with the pattern of celldivision observed for cells of the neurogenic region inthe 14th mitotic cycle (Foe, 1989). The majority of thesecells belong to two mitotic domains (called 'N' and 'M'by Foe, 1989; and see Fig. 6), which complete cycle 14relatively late and do not show synchronous divisions ordiscernible mitotic waves. Instead, these cells divideapparently at random, with a cycle 14 length of 95 minor more for domain N, and 115 min or more for domainM. Therefore, despite the fact that all nuclei wereirradiated simultaneously, the time at which the firstgenetically mutant cell appears will vary, even amongdifferent clones induced in the same animal. The factthat Notch transcription is detectable by blastodermstage (Hartley et al. 1987) supports the idea thatzygotically produced Notch product may be present inthose clones formed relatively late.

Results from later irradiations further support thispicture. The Notch~ clones obtained in the 4h ir-radiation were equal in number to controls, but reducedin size. Those induced at 5h were equal to controls inboth respects, and despite later induction were largerthan the Notch~ clones obtained in earlier irradiations.This argues that the 'mutant' clones were in factcomposed of cells containing sufficient wild-type Notchproduct to complete epidermal development. The in-creasing survival and size of the Notch~ clones observedat later timepoints is consistent with the hypothesis thatearlier Notch~ clones that do survive contain perduringNotch+ product.

Very similar data have been reported for Notch~clones induced in a Minute background in imaginaldisks (Dietrich and Campos-Ortega, 1984). In theirstudy, early irradiations did not produce detectableNotch' clones. Intermediate timepoints yielded Notch'clones that were both smaller and fewer in numbercompared to controls, similar to the data we obtained inthe 3 and 4h timepoints. In both studies, late cloneswere comparable to controls. Unfortunately, we couldnot perform earlier irradiations due to excessive mor-tality of the embryos.

We have considered that there are two feasible, albeitunlikely, alternative explanations for survival of theseclones besides perdurance. It is possible that someregions of the epidermis do not require Notch functionafter neuroblast segregation for subsequent develop-mental decisions. For instance, one could propose thatonly those epidermal cells in regions that later give rise

to PNS (Hartenstein and Campos-Ortega, 1986) requireNotch product following CNS segregation. Some sen-sory organs are formed in the naked cuticle regionswhere our clones appeared (Dambly-Chaudiere andGhysen, 1986), but their small numbers are insufficientto account for the significant loss of clones at 3 h or forthe small size of clones at 4h.

An alternative explanation for the preferential sur-vival of small Notch~ mitotic clones would allow for alimited degree of true nonautonomy such that a mutantcell can form epidermis, but only when surrounded bywild-type epidermal neighbors. Nonautonomy in thisscheme would occur if the Notch protein interactsreciprocally with another component on neighboringepidermal precursor cells, and if the quantitative orarchitectural requirements for the interaction are metonly when a mutant cell is completely surrounded bywild-type neighbors. This interpretation of our resultshas been favored by others (Campos-Ortega, 1990).

The only reason, however, for favoring single cellnonautonomy over perdurance as an explanation forsmall clone survival is the study of Technau andCampos-Ortega (1987). In their experiments, donorcells labelled with HRP were taken from mutantembryos and transplanted into wild-type embryos.There are several caveats that must be borne in mindwhen considering their experiment. First, there was noindependent marker of genotype for the donor em-bryos. That is, they judged whether the donor wasNotch~ by looking at its phenotype at the end ofembryogenesis. The drawback of this criterion is thatdamage to an embryo, such as pricking with a micropip-ette to remove a cell, can produce abnormalities whichmight be confused with a neurogenic phenotype in adonor embryo (Illmensee, 1972). Furthermore, theproportion of donor embryos appearing 'mutant' washigher than expected, as would be predicted if damagewere producing mutant phenocopies in wild-type em-bryos. (In their experiment testing zygotic gene activity,analogous to our mosaic experiment, they saw 14apparent Notch~ embryos and only 28 wild-type,clearly not a 1:3 Mendelian ratio. In another exper-iment, assaying the maternal contribution of Notch, theratio was closer to expectation; J. A. Campos-Ortega,personal communication). A final reservation derivesfrom their whole mount scoring of epidermal pheno-type based solely on the position of HRP-filled cells instage 14 embryos. A cell that had occupied a place onthe blastoderm surface after transplantation, but hadnot otherwise undergone any further development,would not necessarily be an epidermal cell, but mightsuperficially resemble one in the absence of moredetailed morphological criteria.

Cells that have been dissociated and transplanted, onthe other hand, may differ in their developmentalbehavior from clones induced in situ. There is pre-cedent for such an effect in Drosophila, both in im-aginal disk cells (Gehring, 1973) and in mesoderm(Lawrence, 1982, 1987; Beer et al. 1987). Moreover,experiments in amphibian embryos have also suggestedthat isolated cells behave differently from aggregates


when taken out of their normal developmental context(Gurdon, 1988).

Notch as a stabilizer of cell contactsCells that are determined not to be neuroblasts mustform an epithelial sheet with its appropriate adhesive-ness and architecture, clearly differing from that ofneuroblasts. We had suggested earlier that Notch mightbe part of an adhesion system (Hoppe and Greenspan,1986), an idea echoed subsequently by Cagan andReady (1989).

A simple model for Notch's role in maintenance ofepidermal fate would be as a stabilizer of contactsbetween epidermal cells. Such contacts might maintain(and perhaps help to create) the differential adhesionbetween those cells staying on the periphery (epider-mal) and those delaminating inward (neuroblasts). It isinteresting to note in this context that the cytoplasmictail of the large form of N-CAM shows amino acidhomology to Notch's cytoplasmic tail (Barbas et al.1988).

This work was supported in part by NSF grant no. BNS-8616928 to R.J. Greenspan and E. Wieschaus. We thankSpyros Artavanis-Tsakonas, Kristin Johansen and Rick Fehonfor their gift of antibodies to the Notch protein. We offerspecial thanks to Eric Wieschaus for help and suggestionsthroughout this project. We also thank Dan Sweeton forinstruction in embedding and sectioning embryos. In ad-dition, we thank Trudi Schupbach, Eric Wieschaus, IvaGreenwald, Eleanor Maine, Saul Zackson and Dick Horn forhelpful discussions and comments on the manuscript.

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(Accepted 17 May 1990)

notch locus of drosophila is required in epidermal cells ...876 p. e. hoppe and r. j. greenspan...

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