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

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1 Development 109, (1990) Printed in Great Britain The Company of Biologists Limited The Notch locus of Drosophila is required In epidermal cells for epidermal development PAMELA E. HOPPE 1 * and RALPH J. GREENSPAN 2 1 Department of Biology, Princeton University, Princeton, NJ 08544, USA 2 Department 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, MO 63110, USA To whom all correspondence should be addressed Summary The Notch locus of Drosophila plays an important role in cell fate decisions within the neurogenic ectoderm, a role thought to involve interactions at the cell surface. We have assayed the requirement for Notch gene expression in epidermal cells by two kinds of genetic mosaics. First, with gynandromorphs, we removed the wild-type gene long before the critical developmental events to produce large mutant clones. The genotype of cells in large clones was scored by means of an antibody to the Notch protein. Second, using mitotic recombination, we removed the gene at successively later times after completion of the mitotically active early cleavage stages, to produce small clones. These clones were detected by means of a linked mutation of cuticle pattern, armadillo. The results of both experiments demonstrate a requirement for Notch expression by epidermal cells, and thus argue against the model that the Notch product acts as a signal required only in the neuroblast to influence neighboring epidermal cells. The mitotic recombination experiment revealed that Notch product is required by epidermal cells subsequent to neuroblast delamination. This result implies that the Notch gene functions to maintain the determined state of epidermal cells, possibly by mediating cell surface interactions within the epidermis. Key words: neurogenesis, mosaics, determination, Drosophila, Notch, epidermal development. Introduction The Notch locus of Drosophila is required for the proper assignment of developmental fates in ectodermal cells. During embryogenesis its activity is needed for the correct parcelling of cells within the neurogenic ectoderm into neural and epidermal fates. In normal development, the neuroblasts, the precursor stem cells of the embryonic nervous system, arise in a stereotyped pattern from the neurogenic ectoderm located on either side 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 nerve cord. The cells within the neurogenic region that remain at the surface of the embryo become dermatoblasts and give rise to the epidermal layer, which secretes the embryonic cuticle. Embryos homozygous for a deletion of the Notch locus develop a greatly hypertrophied nervous system as a result of the misrouting of epidermal precursors into the neural pathway of development (Poulson, 1940). Similar phenotypes are produced by six other zygotically acting loci (Lehmann etal. 1981; Lehmann et al. 1983). Because deletion of any of the neurogenic loci produces the same neuralized phenotype, and because genetic interactions occur between mutations in different neurogenic loci (Vassin et al. 1985; de la Concha et al. 1988), the products of these loci are believed to be components of a common process that guides cells within the neurogenic ectoderm into the proper developmental pathway. Cell-cell interactions appear to play a central role in the determination of these developmental fates. Ablation studies in the grasshopper have shown that, when a neuroblast is killed, a neighboring undifferentiated ectodermal cell changes its fate to assume that of the absent neuroblast (Doe and Goodman, 1985). This result implies that the neuroblast somehow communicates its presence to surrounding cells and thereby prevents them from becoming neuroblasts. The neurogenic loci of Drosophila may encode components required for the interaction between neuroblast and surrounding cells. Mutations in these genes would thus

2 876 P. E. Hoppe and R. J. Greenspan impair cell communication, allowing epidermal cells to assume the neuroblast fate. The molecular characterization of the locus provides further evidence that Notch plays a role in cell surface interactions. The sequence of the Notch transcript predicted (Wharton et al. 1985; Kidd et al. 1986), and subsequent biochemical studies have identified (Kidd et al. 1989; Johansen et al. 1989), a very large transmembrane protein (2703 amino acids). Because Notch is believed to mediate an interaction between developing cells, it is crucial to define accurately the time and place of its action. To elucidate the role of the Notch product, we wanted to perform a functional test to determine which cells in the neurogenic region required the Notch protein for correct neuroblast segregation. One model is that the Notch product may be required in the neuroblast to signal adjacent cells to become epidermis, but that it is not required in the epidermal cells themselves for proper differentiation. This proposal is supported by cell transplantation experiments in which single cells mutant for Notch were able to form epidermis when transplanted into wild-type hosts (Technau and Campos-Ortega, 1987). However, the alternative view, that expression of Notch is required autonomously by epidermal cells to follow the epidermal pathway, was suggested by the results of a gynandromorph mosaic experiment (Hoppe and Greenspan, 1986). In that experiment, a cuticle marker was used to assess the ability of Notch~ cells to differentiate as epidermis in gynandromorph mosaic embryos. No Notch~ cells produced cuticle within the neurogenic region, indicating that the gene's function was required autonomously by epidermal cells in large clones to complete differentiation. By relying on a cuticle marker scorable only at the end of embryogenesis, it is conceivable that we could have missed local nonautonomy at clonal borders because erosion at the edge of the cuticle would have destroyed the evidence for mutant epidermal cells. To address this possibility, we have now investigated cell fate decisions along borders of large clones at a much earlier stage when we can score epidermal cells directly. Specifically, we have repeated the gynandromorph mosaic experiment, but have used an antibody specific for the Notch protein (Johansen et al. 1989) to distinguish wild-type and mutant cells shortly after neural segregation has occurred. We could thereby assess the ability of Notch' cells to initiate epidermal development by examining cells at mosaic borders early in embryogenesis to determine whether mutant cells had entered the epidermal pathway. To investigate whether epidermal cells require the Notch product after the events of neural segregation have occurred, we have made use of mitotic recombination as an alternate, finer tool to produce mosaics. In this experiment, we induced small clones of Notch~ cells by irradiating embryos heterozygous for a null allelle of the locus. Materials and methods Induction of clones Virgins of the genotypes w arm YD3s N XK "/FM7 and w arm VD35 1FM7 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. After several changes of fresh plates, eggs were collected for 30 min periods, then allowed to develop at 22 C for 3, 4, or 5 h from the midpoint of the collection. The embryos were irradiated for 4 min at approximately 350 radmin" 1, placed at 25 C for approximately 12 h, washed, dechorionated with bleach, and placed in water to complete development (see Gergen and Wieschaus, 1985). Because the frequency of clone induction depends upon the number of cells that are in the appropriate phase of the cell cycle at the time of irradiation, and the size of the clone at the end of development depends upon the number of cell divisions that follow, the survival of the armadillo* Notch~ clones is gauged by comparison to marker {armadillo' alone) clones induced at the same timepoint of irradiation. To control for possible occurrence of false clones, eggs laid by Oregon Rflieswere collected and irradiated in an identical fashion. In addition, several collections from w arm N/FM7 females were processed without being irradiated. To determine the developmental stage at each timepoint of irradiation, a few collections were fixed by the methanol massdevitellinization procedure (Wieschaus and Nusslein-Volhard, 1986), stained with 0.1% toluidine blue O in 20% ethanol for 30 s, dehydrated in ethanol, and mounted in 1:1 Canada balsam: methyl salicylate for examination. Cuticle preparations were made according to Van der Meer (1977). Experimental and control progeny from each time point of irradiation were mounted separately on slides. To prevent any bias in the scoring of clones, the labels on all slides were covered and the slides scrambled prior to examination. The number of clones in each animal and the number of denticles in each clone were recorded. The comparison of Notch~ and control clones via cumulative distribution curves (Fig. 8) required that control values be adjusted to reflect clones expected in a given number of control animals, since the number of control animals scored was never exactly equivalent to the number of experimentals. Thus, the 3h values were multiplied by 245/217 to estimate the number expected in 245 animals rather than the 217 actually scored. Similarly, the 4h controls were multiplied by 146/301 and 5 h controls by 171/205. Antibody staining and sectioning of gynandromorph embryos A rabbit antiserum raised against a fusion protein containing a portion of the Notch coding sequence from the EGF-repeat region was obtained from Kristin Johansen in the laboratory of Spyros Artavanis-Tsakonas. The staining protocol used was the same as reported by Johansen et al. (1989) with the exception that antibody was used at a higher concentration (1:50 in PBS containing 1 % BSA), the Vectastain Elite ABC kit was used to amplify the signal, and development times were longer, up to 1 h. To eliminate nonspecific staining that could appear under these conditions, the antiserum was preabsorbed with mass-devitellenized embryos of 16 h or more of age collected from the N XK "/FM7 stock. These additional steps were necessary to produce No/c/i-specific stain of sufficient intensity to detect in plastic-sectioned embryos. Specificity of the antibody stain in our hands was confirmed in two ways. First, no staining appeared when either primary or secondary antibody was omitted from the

3 Epidermal requirement for Notch 877 procedure. Second, the mosaic-generating cross (described below) produces a significant number of completely Notch' animals, recognizable by their neuralized phenotype, that exhibit no staining in any tissue. In addition, each mosaic animal is in itself a control for Notch specificity because neuralized mutant regions contain nonstaining cells such as dorsal epidermis or mesoderm while wild-type regions of the same animal exhibit staining in these same tissues. Virgins of the genotype y N XK "/FM7 were mated to males carrying the unstable ring-x chromosome, In(l) w vc /N + Y, and placed in collecting chambers (see above) at 25 C. Eggs were collected for 2h periods, then aged for 7h from the endpoint of the collection. Collections were washed, dechorionated in bleach, and fixed in 2 ml of Bouin'sfixative (Sigma) and 4 ml heptane for 15min, transferred to double-stick tape and devitellinized under fresh Bouin's with a sharpened tungsten wire. After removing fixative with washes of PBS, blocking serum (Vector Vectastain ABC Kit) was applied for at least lh. The embryos were incubated with preabsorbed primary antiserum for 5h, rinsed lh (PBS with 1% BSA), incubated with biotinylated secondary antibody (Vectastain Kit) for 2h, rinsed for 30min, placed in ABC reagent (Vectastain Elite Kit) for 45 min, rinsed in 3 changes of PBS with 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 in 0.06% hydrogen peroxide, 0.05% diaminobenzidine (Sigma) in CATbuffer for up to 1 h. Embryos were embedded in Epon 812 and cut in 2 or 3 micron sections as described by Wieschaus and Sweeton (1988). The sections were counterstained with toluidine blue-borax for 10 min on a warm plate, then destained for 15 min (see Altman and Bell, 1973) and airdried prior to applying DPX mountant (Gurr) and a coverslip. 1984). Large numbers of them were fixed, stained and embedded, and after being viewed under a dissecting microscope, seven embryos containing a considerable length of mosaic border within the neurogenic region were selected for cross-sectioning. The sections were examined, and at each point where the border of antibody staining fell within the neurogenic region of the embryonic thorax or abdomen, we scored the morphology of cells on either side as neural or epidermal. All of the epidermal cells along these borders were Notch +. If Notch~ cells had been able to initiate epidermal development, we would have expected to find small, unstained cells that had remained at the embryonic surface and joined the wild-type epidermal layer. However, despite the examination of more than 200 border points sampling over 100 border epidermal cells, not one Notch~ epidermal cell was found (Fig. 2). To maximize the possibility of detecting rare, mutant epidermal cells, we limited our analysis to those borders most likely to harbor such a cell. This excluded the large number of mosaic borders falling at the ventral midline, due to the prior invagination of mesodermal and mesectodermal cells at this point. Because cell-cell interactions may not occur between cells on either side of the midline, a mosaic border at the ventral midline may not test the autonomy of Notch gene function. In addition, we excluded borders that fell in the most dorsal area of the neurogenic region, due to the A. Notch is required only In the neuroblast Results Epidermal cells along gynandromorph borders always express Notch If Notch expression is required only by neuroblasts as a signalling protein, then mutant epidermal cells should be present at gynandromorph borders (Fig. 1). To ascertain the ability of mutant cells along the border of a large Notch~ clone to enter the epidermal pathway, we used an unstable ring-a' chromosome to produce mosaics containing large areas of mutant cells. The genotype of the cells was assessed by staining the mosaics with an antibody specific for the Notch protein (Johansen et al. 1989; also see Methods). Since the Notch product is a transmembrane protein bound to the cell, the antibody serves as a cell autonomous marker. Neuroblasts and epidermal cells were distinguished by morphological criteria in plastic sections. As neuroblasts delaminate, they enlarge, become round and begin dividing, producing smaller daughters dorsally. In contrast, epidermal cells are small, divide within the plane of the embryonic surface and initially adopt a more rectangular shape within the epithelium before becoming quite flat, spreading over the embryo's surface (Poulson, 1950; Campos-Ortega and Hartenstein, 1985). The animals were permitted to develop for at least 7 h, at which time the process of neuroblast segregation is virtually complete (Hartenstein and Campos-Ortega, V. mutant cellsremainin epidermis when In contact with a wt nauroblast B. Notch is required autonomously by epidermal cells ALL mutant c*ta become neurobtasts Fig. 1. The configuration of mutant and wild-type (wt) cells at mosaic borders tests the autonomy of Notch action in the epidermis. (A) If the Notch protein is required only by the neuroblast to signal adjacent prospective epidermal cells, some mosaic borders will appose a wild-type neuroblast and mutant prospective epidermal cells, resulting in Notch~ cells differentiating as epidermis at the mosaic border. (B) If epidermal cells in the neurogenic region require Notch product autonomously, then all mutant cells at all mosaic borders will become neuroblasts. Small ellipses represent prospective epidermal cells, large circles represent neuroblasts and shading indicates mutant genotype.

4 878 P. E. Hoppe and R. J. Greenspan difficulty in precisely locating its dorsal limit. Outside of this limit, epidermal differentiation is not dependent on the action of the Notch locus (Lehmann et al. 1981; Hoppe and Greenspan, 1986). Our failure to find any mutant epidermal cells in the neurogenic region indicates that in large clones each epidermal cell, even those along the mosaic border, must express the Notch protein. Among the more than 200 borders scored, it was not possible to determine directly at any given border point whether a Notch + neuroblast was ever present where it might have permitted a Notch~ epidermal cell to survive. We were unable to score neuroblast genotype directly because, although all cells in the neurogenic region initially stain with antibody during neuroblast segregation (Kidd etal. 1989; Johansen etal. 1989; and our unpublished observations), at the time neuroblast segregation is complete even wild-type neuroblasts fail to stain (Fig. 3). (The lack of staining in neuroblasts at this later time could probably not be discerned in whole mount preparations because of surface epidermal staining. Antibody staining of neurons is evident in later stages, particularly along their axons; Kidd et al. 1989; Johansen et al. 1989; and our unpublished observations.) However, given the separation of neural and epidermal lineages, as well as the randomness of mosaic borders, if it were possible for a mutant cell to differentiate as epidermis under the influence of an adjacent wild-type neuroblast, it is virtually certain that we would have seen it among the more than 100 epidermal cells sampled. The failure to find separation between Notch + genotype and epidermal phenotype in over 100 cells means that if the genotype of some other cell was critical, that cell must map <1 sturt from the epidermal cell. Adjacent cells in the blastoderm map at least 2-3 sturts apart (Szabad et al. 1979), so it is unlikely that the genotype of some other cell (e.g. a neuroblast) is important. [The sturt is a measure of developmental relatedness based on the probability of a mosaic border passing between any two points on the blastoderm. It is conceptually related to a genetic map unit, and is similarly calculated (Garcia-Bellido and Merriam, 1969).] This analysis of mosaic borders in gynandromorphs shortly after neural segregation confirmed the autonomy of Notch in epidermis observed in cuticle preparations (Hoppe and Greenspan, 1986). The previous mosaic study, however, had indicated that in addition to the lack of mutant cuticle, a loss of wild-type cuticle occurred prior to the end of embryogenesis. The clearest indication of this loss was the lack of isolated 'islands' or 'peninsulas' of wild-type cuticle in mosaic animals. It was unknown whether the deficit of wildtype cuticle was the result of the misrouting of wild-type cells at the time of neural segregation, of a later failure of differentiation, similar to that described for dorsal epidermal cells in neurogenic mutants (Jimenez and Campos-Ortega, 1982), or of actual erosion of cuticle. Examination of the mosaic borders in antibodystained sections allowed us to detect certain configurations 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. A number of the sections contained small ventral patches of wild-type epidermal cells not connected to the dorsal region of epidermis unaffected in Notch~ embryos (Fig. 4). The observation that isolated patches of wildtype cells can initiate epidermal development suggests that our previous report of a loss of wild-type cuticle in mosaics (Hoppe and Greenspan, 1986) is a later, secondary degenerative event, rather than the result of the misrouting of wild-type cells during neural segregation. Moreover, the presence of epidermal islands in the preparations argues that our failure to detect Notch~ epidermal cells at borders is not due to loss of these cells following segregation. Notch product is required in epidermal cells after neuroblast delamination To investigate further the role of the Notch gene in the epidermis, we induced clones consisting exclusively of epidermal cells later in development by mitotic recombination (Fig. 5). Therefore any effect of the Notch mutation in these clones, none of which are associated with a mutant neuroblast, cannot be the result of the Notch product functioning solely as a signal from neuroblasts to epidermal cells. For detection of mitotic clones in the epidermis, the Notch mutation was linked to a null allele oiarmadillo, a cell autonomous 'segment polarity' gene (Nusslein-Volhard and Wieschaus, 1980; Wieschaus et al. 1984; Wieschaus and Riggleman, 1987). We determined that embryos mutant for both genes exhibited the neuralized phenotype characteristic of Notch~ embryos, as to be expected since there is no epidermis to form denticles. Because of their close linkage, any induced Notch~ clones would also be homozygous for the armadillo mutation. Such clones would produce clusters of ectopic denticles in regions of naked cuticle, but only if genotypically Notch~ cells can survive and secrete cuticle. Synchronously aged collections of embryos were irradiated at several different timepoints. Irradiations performed during cleavage stages resulted in aborted development in the majority of embryos. The first successful irradiations were at the 3h timepoint, when virtually all embryos (61/64) were in the process of cellularization, embryonic stage 5 (Campos-Ortega and Hartenstein, 1985; Wieschaus and Nusslein-Volhard, 1986; and see Fig. 6), as ascertained by examination of fixed collections (see Methods). At this age all blastoderm nuclei are in interphase of mitotic cycle 14 (Foe, 1989; and see Fig. 6). Therefore, recombination events induced at this time result in the production of one mutant daughter, the first homozygous Notch~ cell, upon completion of the 14 th mitosis. Since the majority of neuroblasts delaminate prior to the completion of cycle 14 (Hartenstein and Campos-Ortega, 1984), the tuning of this division and the fact that only one daughter cell can be mutant means that clones will be restricted to either the neuroblast or the epidermal lineage. Fig. 5 illustrates the possible configurations that produce this result. Because the armadillo marker

5 ms WT vm Fig. 2. Examination of mosaic borders revealed no unstained (mutant) cells in the epidermal layer. In A, a mutant patch lies on one side of the ventral midline (vm). The wild-type epidermal cell at the mosaic border is indicated by the small arrow. To the right, this cell is adjacent to wildtype epidermal cells that form a smooth epithelium of small cells with small nuclei. To the left of this border cell, the external outline of the embryo becomes irregular, and the unstained cells, indicated by arrowheads, are large cells with large nuclei that have the rounded appearance of neuroblasts. No unstained cells have joined the epidermal layer at its border. In B, a nearby section is shown where, again, the wild-type epidermal cell at the mosaic border is indicated by the small arrow. The unstained cells at the mosaic border, indicated by arrowheads, have not joined the smooth epidermal epithelium, and like neuroblasts are dividing to produce daughters dorsally (interiorly). Fig. 3. Neuroblasts do not stain with the Notch antibody after neural segregation is complete. A section of a Notch mosaic where involution at the ventral furrow during gastrulation caused the gynandromorph border to fall at the ventral midline (vm) of the ectodermal anlagen is shown. All ectodermal cells on the right (N~) side fail to stain with the Notch antibody, although mesodermal (ms) staining demonstrates sufficient permeability to the antibody. On the wild-type (WT) side, the epidermal layer of cells continues to stain with the antibody (small arrows) but the neuroblasts (arrowheads) do not. The staining detects Notch protein in the cytoplasm as well as on the surface of epidermal cells, which contain significant Notch transcript at this time (Hartley el al. 1987); this is probably due to the measures taken to intensify the antibody signal (see Methods). Fig. 4. A small island of wild-type cells is able to initiate epidermal development. In the section shown, involution at the ventral furrow during gastrulation has produced a small group of wild-type ectodermal cells at the ventral midline, and a few cells (small arrows) have initiated epidermal development. Mesodermal staining (ms) is also evident.

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7 Epidermal requirement for Notch 879 A NB SEG DIV B. NB DIV NB c DIV SECOND NB SEG ^ Fig. 5. Irradiation in cycle 14 can produce mutant clones that contain epidermal cells or neural cells, but not both. In each case 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 is a prospective epidermal cell (indicated by ellipses); any mutant progeny that survive in the epidermis will be detected by the armadillo marker. In B, the mutant cell is produced upon the first division of the NB, generating either a mutant NB or a mutant ganglion mother cell. Neither of the events in B are detected by the armadillo marker; thus our experiment does not test the effect of Notch in strictly neural clones. In C, the target cell completes cycle 14 on the blastoderm surface before the end of neuroblast segregation. The one mutant daughter will either become a NB, an event not detected by the armadillo marker, or initiate epidermal development, in which case any mutant progeny that differentiate in the epidermis will be detected by the armadillo marker. In no case is a marked (mutant) epidermal cell adjacent to a mutant NB. Therefore, our experiment tests the effect of the Notch mutation on the development of strictly epidermal clones. detects mutant epidermal cells only, recombination events in the neural lineage do not produce detectable clones and could not be studied (Fig. 5B). Therefore, our study is restricted to small epidermal Notch~ clones that develop in the context of a fully wild-type complement of neuroblasts (Fig. 5A,C). Even in the case where a neuroblast delaminates only after completing cycle 14 on the embryonic surface (Technau and Campos-Ortega, 1986), the mutant clone produced will contain exclusively neural or epidermal cells (Fig. 5C). Any effect of the Notch mutation on the number and size of these clones would have to reflect a requirement for Notch product in the epidermis. The timing of mitoses in this cell population also means that virtually all neuroblasts will have delaminated by the time clones become homozygous. They will have left the blastoderm layer in the SI and S2 waves (see Fig. 6 and also Hartenstein and Campos- Ortega, 1984 and Foe, 1989), which precede the earliest possible homozygosing of clones. Thus, any effect of the Notch mutation on induced clones would also reflect an epidermal role for the gene product subsequent to any interactions with presumptive neuroblasts. Irradiation at the 3 h timepoint resulted in a very high frequency of armadillo' clones in control animals (0.48 clones/total progeny), one or more clones being present in virtually every heterozygous animal (Table 1). Some clones were also identified in experimental animals, indicating that a few genotypically armadillo' Notch cells were able to complete epidermal development and secrete cuticle (Fig. 7). However, comparison of the experimental and control clones revealed that the number of Notch~ clones scored was sharply lower (frequency=0.21), indicating that just over half of the expected clones failed to differentiate in the epidermis. In addition, the Notch~ clones that did appear were smaller, judging from the average number of denticles produced (4.5 denticles/ clone), compared to controls (6.4 denticles/clone). Denticle morphology and density were similar in experimental and control clones. The predominance of small clones in the Notch~ sample is evident in a comparison of the cumulative distribution curves of experimental and control clones (Fig. 8A). Since each epidermal cell is thought to secrete about 3 denticles (Wieschaus and Riggleman, 1987), most of the surviving Notch' clones (80%) are one or two cells in size. The 4h timepoint contained embryos in early gastrulation, stages 6, 7 and 8. At this time the cells of the neurogenic region are still in cycle 14 (Foe, 1989). In controls, the irradiation at this time produced substantially fewer clones than controls at 3h, but they were still the same size (Table 1, Fig. 8B). This suggests that, although no cell division has occurred, the number of cells that are targets of mitotic recombination is lower at 4h than at 3h, but that those remaining responsive will undergo the same amount of proliferation. Notch~ clones, in contrast, appeared at a frequency equal to controls (0.14 and 0.16, respectively), but were signifi-

8 880 P. E. Hoppe and R. J. Greenspan Irradiations 3 hr 4 hr 5hr earliest possible homozygous clones clones missing and small clones small clones normal ectoderm appears 'neurogenlc' S1 nb's delam mitoses domaln mitoses hrat22 C 0 1 S2 nb's delam., S3 nb's delam. 10 Stages 10 % X \\ ^\ V cephalic furrow 9> o^ appears \ \ Fig. 6. Time course of embryogenesis with reference to Notch clone induction, neuroblast delamination and mitosis in the neurogenic region. SI, S2 and S3 refer to waves of neuroblast delamination, SI constituting 61 % and S2 constituting 21 % of the total (Campos-Ortega and Hartenstein, 1985). Thus, 82% of the neuroblasts have delaminated by the time the first mitoses begin in the neurogenic region ('N' and 'M' domains of Foe, 1989). Timepoint of irradiation 3 h cycle 14 blastoderm Table 1. Notch and control clones marked with armadillo control Notch' Number of clones Clone frequency Clone size S.E. (0.42) (0.48) 4 h cycle 14 early gastrula control Notch' (0.58) (0.64) 5 h cycle stomodeal invag. control Notch' (0.54) (0.48) Clone frequency represents the number of clones per total animals scored. Clone size is expressed in average number of denticles per clone. s.e. denotes standard error of the mean. * Notch' clones significantly smaller than controls, Mest P= ** Notch' clones significantly smaller than controls, f-test / ) = cantly smaller on average (4.4 vs. 6.2 denticles/clone) (Table 1, Fig. 8B). This indicates that, although the expected number of Notch~ clones survive at this time point, the cells completed fewer divisions or some members of the clone were lost. The 5h collection spanned the time of stomodeal invagination, containing stage 9 and 10 embryos. Irradiation at this time could result in recombination in cells still in cycle 14 or in cells that have divided and entered cycle 15. Notch and control clones induced at this stage were indistinguishable in frequency and size (Fig. 8C). The similarity in behavior of Notch~ cells and control cells suggests that sufficient zygotic expression of the gene has occurred by the time they divide. The fact that irradiations at these three different times give different results, even though they all precede cell divisions (see Fig. 6), conforms with the observed asynchrony of mitosis in this region during

9 Epidermal requirement for Notch 881 ** A A 3hr 80- * \ "»> \»'",,'.,».< en B 4hr a> n E 40- a> > 20- arm N < - E u c 5hr arm N Fig. 7. /Vofc/i and control clones induced at 3 h and marked with armadillo result in the appearance of ectopic denticles in regions of naked cuticle. In A, the arrow indicates a Notch' clone comprising 5 denticles. In B, the arrow indicates a control clone comprising 15 denticles. cycle 14 (Foe, 1989) and with differential susceptibility of cells to mitotic recombination due to their place in the cell cycle. Discussion The product of the Notch locus takes part in the specification of embryonic cell fate through its expression by presumptive epidermal cells. The time course of its requirement by these cells argues that it is involved in the maintenance of the determined epidermal state. These two conclusions follow from analysis of mosaic experiments presented here. In conjunction with what is known about the structure of the gene product, these results suggest that the Notch protein may act by stabilizing contacts between cells in the epithelial sheet of epidermal precursors. Notch expression is required in epidermal precursors Two kinds of mosaic analysis have been carried out to , r =J-i M~^ arm Size of Clone (number of denticles) Fig. 8. Cumulative distributions of clones. In each graph, cumulative distribution curves are used to compare the size distribution of Notch~ and control clones obtained at a given timepoint. Because the number of clones depends upon the number of animals scored, the comparisons were made by drawing the true distribution of Notch~ clones and adjusting the control values to reflect the clones expected in a control population exactly the same size as the experimental population (see Methods). (A) Irradiation at 3 h produces fewer Notch~ clones in virtually all size classes, as shown by the difference in the height of the two curves. Those Notch~ clones that are obtained are also smaller, the curve reaching nearly maximum height earlier, in the small clone sizes. (B) Irradiation at 4h produces equal numbers of clones, but, as reflected in the faster rise of the Notch' curve, the Notch~ clones are smaller (see also Table 1). (C) Irradiation at 5h produces Notch' clones that are indistinguishable from controls. determine which cells require Notch expression for correct specification of cell fate. When assayed with an antibody to the Notch protein, large clones in gynandromorphs show a strict correlation between Notch + genotype and proper epidermal development. To score accurately as many border cells as possible, we re-

10 882 P. E. Hoppe and R. J. Greenspan covered sections and reconstructed the relevant clone boundaries from seven mosaics. If the genotype of some other cell were crucial, we would have detected some Notch~ epidermal cells at these borders. In mitotic recombination experiments, over half of the expected epidermal clones induced by early irradiation fail to appear, and those that are recovered are smaller than controls. The timing of cell division in the neurogenic region is such that these mitotic clones are restricted to the epidermal lineage (Fig. 5). The reduction in epidermal clone number and size cannot, therefore, be attributed to the genotype of any other cell type. Both experiments concur that wild-type gene product must be expressed by the cells that will become epidermis. They are consistent with recent mosaic findings concerning the neural-epidermal decision in imaginal discs, demonstrating a requirement for Notch product in cells choosing the epidermal fate (P. Heitzler and P. Simpson, personal communication). Moreover, all of these experiments contradict a model ascribing the site of Notch gene action exclusively to neuroblasts (Technau and Campos-Ortega, 1987; Campos-Ortega, 1988). Our results are also in good agreement with comparable mosaic experiments done in C. elegans with two genes homologous to Notch. The genes lin-12 and glp-1 are known to be required for cell fate decisions in which cell interactions play an important role (Kimble, 1981; Greenwald et al. 1983; Austin and Kimble, 1987; Preiss etal. 1987; Seydoux and Greenwald, 1989). Both lin-12 and glp-1 are transmembrane proteins that contain portions of both the intracellular and extracellular domains 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 be required autonomously in the VU cell for VU development (Seydoux and Greenwald, 1989), while glp-1 was found to act in the germ line for regulation of the germ line 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 discussed by Seydoux and Greenwald (1989) was that in mosaic worms containing one mutant and one wild-type cell in the interacting pair, the mutant cell always became the anchor cell. One possibility they raised was that initially there is symmetrical expression of lin-12 in both cells, which eventually tilts towards a preponderance of expression by the VU cell and a reciprocal reduction in the anchor cell. This idea has received some recent support in Drosophila from mosaic experiments in imaginal discs employing duplications of the Notch locus (P. Heitzler and P. Simpson, personal communication). In these experiments, cells containing more copies of wild-type Notch preferentially adopted the epidermal fate. Because neuroblasts originally stain for Notch, but lose staining at least transiently after segregation (Fig. 3), one can speculate that the disappearance of Notch activity in neuroblasts may be as important as its persistence in epidermal cells to specify correct cell fate. Notch is required beyond the time of neuroblast delamination The fact that our mitotic clones do not lose their wildtype Notch allele until after neuroblast delamination is virtually complete (Fig. 6) provides us with new information on the time course of the gene's action. It argues that there is an ongoing requirement for Notch gene product in epidermal cells for a period of time following the initial interactions between equivalent cells in the neurogenic ectoderm. This can best be thought of as a role in maintaining the determined state. There is already good reason to believe that the neural-epidermal decision requires stabilization. Heterochronic transplants of ectodermal cells between neurogenic and non-neurogenic regions demonstrate that even after neuroblast delamination, a presumptive epidermal cell can change fate and become a neuroblast (Technau et al. 1988). Similarly, temperature-shift experiments with the mutant shibire" have defined a longterm requirement for this gene's activity in normal neurogenesis, extending past the end of neuroblast segregation (Poodry, 1990). Our experiments support the idea that Notch mediates such a stabilization, since the removal of Notch* activity in presumptive epidermal cells, even after the vast majority of neuroblasts have delaminated (Fig. 6), results in loss of these cells from the epidermis. Moreover, Notch is known to act at multiple times in development. Studies of a temperature-sensitive allele and of mitotic clones induced in imaginal disks have demonstrated a requirement in later stages of embryonic (Shellenbarger and Mohler, 1978), and larval development (Portin, 1980; Dietrich and Campos-Ortega, 1984; Cagan and Ready, 1989). Analysis of the appearance of Notch transcript and protein has also revealed that it is expressed at later stages of development and continues to be present in epidermal cells after the completion of central neurogenesis (Fig. 3; Hartley et al. 1987; Kidd et al. 1989; Johansen et al. 1989). Whether Notch is also involved in the initiation as well as the maintenance of the determined state in epidermal cells remains an open question. None of the mosaic experiments have been able to address this issue. In the case of lin-12, mosaic experiments did provide evidence for a role in initiation of the determined state of the VU cell. In all of the mosaics examined, Seydoux and Greenwald (1989) found that whenever one cell of the pair was mutant, it always became the anchor cell and the other always became a VU cell. If the gene were required only for maintenance, and initiation occurred independently, then it should have been possible to obtain mosaics in which only one cell was mutant but both were anchor cells. The available evidence for Notch and lin-12 is compatible with both genes playing roles in initiation and maintenance of the determined state.

11 Epidermal requirement for Notch 883 Survival of some genotypically Notch clones is due to perdurance A question remains as to why some of our Notch~ clones do survive after irradiation at the earliest time point. The most likely explanation is perdurance of Notch + product in genotypically mutant cells. Because large clones probably derive from mutant precursor cells that were both established early, prior to significant zygotic Notch expression, and subject to a greater number of subsequent cell divisions, which further dilute the product, cells of a large clone would be less likely to contain sufficient Notch product to complete epidermal development. This explanation conforms with the pattern of cell division observed for cells of the neurogenic region in the 14 th mitotic cycle (Foe, 1989). The majority of these cells belong to two mitotic domains (called 'N' and 'M' by Foe, 1989; and see Fig. 6), which complete cycle 14 relatively late and do not show synchronous divisions or discernible mitotic waves. Instead, these cells divide apparently at random, with a cycle 14 length of 95 min or more for domain N, and 115 min or more for domain M. Therefore, despite the fact that all nuclei were irradiated simultaneously, the time at which the first genetically mutant cell appears will vary, even among different clones induced in the same animal. The fact that Notch transcription is detectable by blastoderm stage (Hartley et al. 1987) supports the idea that zygotically produced Notch product may be present in those clones formed relatively late. Results from later irradiations further support this picture. The Notch~ clones obtained in the 4h irradiation were equal in number to controls, but reduced in size. Those induced at 5h were equal to controls in both respects, and despite later induction were larger than the Notch~ clones obtained in earlier irradiations. This argues that the 'mutant' clones were in fact composed of cells containing sufficient wild-type Notch product to complete epidermal development. The increasing survival and size of the Notch~ clones observed at later timepoints is consistent with the hypothesis that earlier Notch~ clones that do survive contain perduring Notch + product. Very similar data have been reported for Notch~ clones induced in a Minute background in imaginal disks (Dietrich and Campos-Ortega, 1984). In their study, early irradiations did not produce detectable Notch' clones. Intermediate timepoints yielded Notch' clones that were both smaller and fewer in number compared to controls, similar to the data we obtained in the 3 and 4h timepoints. In both studies, late clones were comparable to controls. Unfortunately, we could not perform earlier irradiations due to excessive mortality of the embryos. We have considered that there are two feasible, albeit unlikely, alternative explanations for survival of these clones besides perdurance. It is possible that some regions of the epidermis do not require Notch function after neuroblast segregation for subsequent developmental decisions. For instance, one could propose that only those epidermal cells in regions that later give rise to PNS (Hartenstein and Campos-Ortega, 1986) require Notch product following CNS segregation. Some sensory organs are formed in the naked cuticle regions where our clones appeared (Dambly-Chaudiere and Ghysen, 1986), but their small numbers are insufficient to account for the significant loss of clones at 3 h or for the small size of clones at 4h. An alternative explanation for the preferential survival of small Notch~ mitotic clones would allow for a limited degree of true nonautonomy such that a mutant cell can form epidermis, but only when surrounded by wild-type epidermal neighbors. Nonautonomy in this scheme would occur if the Notch protein interacts reciprocally with another component on neighboring epidermal precursor cells, and if the quantitative or architectural requirements for the interaction are met only when a mutant cell is completely surrounded by wild-type neighbors. This interpretation of our results has been favored by others (Campos-Ortega, 1990). The only reason, however, for favoring single cell nonautonomy over perdurance as an explanation for small clone survival is the study of Technau and Campos-Ortega (1987). In their experiments, donor cells labelled with HRP were taken from mutant embryos and transplanted into wild-type embryos. There are several caveats that must be borne in mind when considering their experiment. First, there was no independent marker of genotype for the donor embryos. That is, they judged whether the donor was Notch~ by looking at its phenotype at the end of embryogenesis. The drawback of this criterion is that damage to an embryo, such as pricking with a micropipette to remove a cell, can produce abnormalities which might be confused with a neurogenic phenotype in a donor embryo (Illmensee, 1972). Furthermore, the proportion of donor embryos appearing 'mutant' was higher than expected, as would be predicted if damage were producing mutant phenocopies in wild-type embryos. (In their experiment testing zygotic gene activity, analogous to our mosaic experiment, they saw 14 apparent Notch~ embryos and only 28 wild-type, clearly not a 1:3 Mendelian ratio. In another experiment, assaying the maternal contribution of Notch, the ratio was closer to expectation; J. A. Campos-Ortega, personal communication). A final reservation derives from their whole mount scoring of epidermal phenotype based solely on the position of HRP-filled cells in stage 14 embryos. A cell that had occupied a place on the blastoderm surface after transplantation, but had not otherwise undergone any further development, would not necessarily be an epidermal cell, but might superficially resemble one in the absence of more detailed morphological criteria. Cells that have been dissociated and transplanted, on the other hand, may differ in their developmental behavior from clones induced in situ. There is precedent for such an effect in Drosophila, both in imaginal disk cells (Gehring, 1973) and in mesoderm (Lawrence, 1982, 1987; Beer et al. 1987). Moreover, experiments in amphibian embryos have also suggested that isolated cells behave differently from aggregates

12 884 P. E. Hoppe and R. J. Greenspan when taken out of their normal developmental context (Gurdon, 1988). Notch as a stabilizer of cell contacts Cells that are determined not to be neuroblasts must form an epithelial sheet with its appropriate adhesiveness and architecture, clearly differing from that of neuroblasts. We had suggested earlier that Notch might be part of an adhesion system (Hoppe and Greenspan, 1986), an idea echoed subsequently by Cagan and Ready (1989). A simple model for Notch's role in maintenance of epidermal fate would be as a stabilizer of contacts between epidermal cells. Such contacts might maintain (and perhaps help to create) the differential adhesion between those cells staying on the periphery (epidermal) and those delaminating inward (neuroblasts). It is interesting to note in this context that the cytoplasmic tail of the large form of N-CAM shows amino acid homology to Notch's cytoplasmic tail (Barbas et al. 1988). 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