We have stained the ovaries of nearly 600 different Drosophila strains carrying single copies of a P-element enhancer detector. This transposon detects neighbouring genomic transcriptional regulatory sequences by means of a β-galactosidase reporter gene. Numerous strains are stained in specific cells and at specific stages of oogenesis and provide useful ovarian markers for cell types that in some cases have not previously been recognized by morphological criteria. Since recent data have suggested that a substantial number of the regulatory elements detected by enhancer detection control neighbouring genes, we discuss the implications of our results concerning ovarian gene expression patterns in Drosophila. We have also identified a small number of insertion-linked recessive mutants that are sterile or lead to ovarian defects. We observe a strong correlation with specific germ line staining patterns in these strains, suggesting that certain patterns are more likely to be associated with female sterile genes than others. On the basis of our results, we suggest new strategies, which are not primarily based on the generation of mutants, to screen for and isolate female sterile genes.

One important aim of contemporary studies of developmental biology is to elucidate the complex mechanisms controlling the development of the oocyte. In the fruitfly Drosophila melanogaster oogenesis is being studied extensively at the cellular, genetic and molecular levels (for review see Mahowald and Hardy, 1985). Several genetic screens have been carried out in Drosophila to isolate female sterile (fs) mutations (e.g. Bakken, 1973; Gans et al. 1975; Mohler, 1977; Komito-poulou et al. 1983; Schiipbach and Wieschaus, 1989). As a result, many fs genes have been identified and partially characterized. These fs mutations can be subdivided into two classes: first, those that lead to abortive egg-production or to abnormal eggs that cannot be fertilized and, second, the fs and maternal effect mutations which lead to the production of mature eggs that can be fertilized but do not develop normally. In the past few years, the roles of some fs genes have been largely defined. For example, genetic screens for maternal effect mutations that alter the cuticular phenotype of first instar larvae have led to the identification of a number of fs genes that are involved in the establishment of the anteroposterior and dorsoventral polarities of the egg (e.g. Anderson et al. 1985; Frohnhöfer and Niisslein-Volhard, 1986; Schüpbach and Wieschaus, 1986; Niisslein-Volhard et al. 1987). Several of these genes have been cloned and it is now beginning to be possible to construct molecular models for the processes that lead to the establishment of the coordinate axes in the oocyte and embryo (e.g. Driever and Niisslein-Volhard, 1988; Sprenger et al. 1989). However, it has proven to be difficult to assign a functional role to the majority of fs genes, since the associated mutants do not lead to an easily interpretable phenotype in either ovaries or deposited eggs. In these cases, it is important to devise methods that, for example, allow convenient identification of cells that are affected by the mutated gene or reveal when and where the mutated gene is normally expressed.

In addition to the problems of assessing the role of fs genes, it is also clear that not all genes required for the production of normal eggs can be recognized by classical genetic screens. It has been shown that most genes involved in oogenesis are also required at other times and in other tissues during development (see Perrimon et al. 1986). If any such gene is essential for development, flies lacking this gene will die and their fertility cannot be assessed. In genetic screens for fs mutants, these essential genes can only be identified by hypomor-phic alleles that affect oogenesis, but do not cause lethality; in many cases, it now appears that such alleles have not been isolated in screens for fs mutations (see Perrimon et al. 1986 and 1989). Germ line dependence for essential genes can be demonstrated by generating germ line clones homozygous for the associated lethal mutation (Perrimon and Gans, 1983; Perrimon et al. 1989). However, this method is rather complicated and only discriminates between germ line and soma, but does not yield more precise information about the cell types that are affected.

Another group of fs genes that cannot be identified in a simple genetic screen consists of genes whose activities are redundant in the sense that they can be complemented by other genes with related functions. In these cases, a mutant phenotype may only be observed when all or most of the related genes are defective in the same organism. A comprehensive study of female sterile genes in Drosophila therefore requires alternative methods that are not primarily based on the recognition of a mutant phenotype.

In this paper, we have analyzed Drosophila oogenesis using the recently developed method of P-element-mediated enhancer detection (O’Kane and Gehring, 1987; Bellen et al. 1989). In this technique, a P-transposon construct containing a β-galactosidase reporter gene fusion is randomly integrated at different sites in the Drosophila genome, where the expression of the reporter gene is influenced by nearby regulatory sequences such as enhancers (O’Kane and Gehring, 1987). A staggering array of spatially and temporally regulated β-galactosidase-staining patterns has been observed in embryos carrying single copies of the P-transposon at different locations (Bellen et al. 1989). Since genomic sequences adjacent to the construct can be rapidly cloned and analyzed in these insertion strains (which are referred to as transposants), it has been possible to show that a significant proportion of the regulatory elements that are active in embryos control nearby Drosophila genes (Wilson et al. 1989). The variety of staining patterns observed in different strains at any stage of development may therefore be considered to reflect the general range of expression patterns of Drosophila genes.

Fasano and Kerridge (1988) have recently stained the adult ovaries of a number of strains containing our enhancer detector, P[lac, ry+] (O’Kane and Gehring, 1987). We have been performing a more extensive analysis of about 600 transposants carrying single copies of the more versatile enhancer detector, P[lArB] (Bellen et al. 1989; Wilson et al. 1989). In addition to many different transposants that can be used as ovarian cell markers, we have identified some novel staining patterns that reveal differences in transcriptional activity between morphologically indistinguishable cells in the soma or in the germ line. Since it is now clear that many of the observed patterns probably represent the expression patterns of neighbouring genes, we have performed a detailed analysis of temporal and spatial expression in all transposants; we compare and contrast our data with previous information on ovarian gene expression in Drosophila derived from genetic studies. Data concerning a small number of insertion-linked sterile and semisterile mutants recovered in our screen suggest that specific ovarian staining patterns may be associated with insertions that disrupt fs genes. In the light of our results, we suggest ways in which P-element-mediated enhancer detection can be coupled with more classical genetic approaches to extend previous studies of genes involved in Drosophila oogenesis.

Detection of β-galactosidase activity in ovaries

Four-day-old females were anaesthetized, washed in ethanol and dissected in BSS (55mM-NaCl, 40mM-KCl, 15 mM-MgSO4, 5mM-CaC12-H2O, l·79g 1“1 tricin, 3·6gl·1 glucose, 17·1 gl—1 saccharose, 1 gl—1 BSA). The ovaries were transferred to microtitre plates containing 30 μl devitellinizing buffer (1 volume buffer B described in Freeman et al. (1986), 4 volumes H2O, 1 volume of 37% formaldehyde) and 200 μl heptane. The microtitre plates were shaken gently for 10 min at room temperature. The devitellinizing buffer was replaced by 300 μl fixation buffer (1% glutaraldehyde in PBS) and incubated for 10 min. The ovaries were washed once with Ringer’s solution and incubated overnight at 37°C in staining solution (Simon et al. 1985) with 0·3 % Triton X-100. The ovaries were dehydrated in series of ethanol solutions of increasing concentration, mounted, viewed with Nomarksi optics and photographed with a Kodak Ektachrome 50 slide film at 25 ASA.

Detection of β-galactosidase activity in ovarian sections

Four-day-old flies were anaesthetized and rinsed in ethanol and water. The flies were transferred into a drop of OCT 4583 medium (Tissue-Tek, Miles) for 5 to 10 min and frozen in liquid nitrogen. Sections were prepared as described by Hafen and Levine (1986), but 1% glutaraldehyde was used as a fixative instead of paraformaldehyde. The slides were washed three times in PBS for 10 min and rinsed in staining buffer (without X-gal). 200 μl staining solution was layered on the preparations, which were covered with a coverslip and incubated overnight at 37 °C. Slides were washed with PBS and sections were counterstained with eosin and mounted in DAKO glycergel.

Remobilizing P[lArB] in transposants

To ascertain whether a mutant phenotype is caused by a P[lArB] insertion, the P[lArB] transposon was excised in order to produce revenants using flies carrying a genomic source of transposase (Robertson et al. 1988). However, instead of performing direct brother and sister matings with the progeny of a jumpstart male to test for revenants (Bellen et al. 1989), single rosy1 progeny of jumpstart males carrying the Δ1P[lArB] chromosome were used to generate heterozygous offspring which were then mated to each other. For example, for the third chromosome insertions the following crosses were performed:
formula

Recovery of homozygous ry506 Δ1P[lArB] females with normal ovaries indicates that the PflArB] insertion is responsible for the mutant phenotype provided no other P-element insertions are present in this strain; no additional insertions have been found in the transposant strains used (see Bellen et al. 1989). Similar crosses were performed for strains with P[lArB] insertions on the first chromosome. To study the morphological characteristics of ovaries of putative revenants, ovaries were dissected and stained with a nuclear dye as described by Bellen and Kiger (1988).

Fertility assays

All assays were performed at 25°C. Individual 1- to 2-day-old homozygous (or heterozygous fs mutant) virgin females were mated to two 1- to 4-day-old Oregon-R males in 8 dram vials containing standard fly food. Flies were discarded after 7 days and progeny were counted at day 18. For strains with decreased fertility, the average progeny of 10 individual females was compared with the average progeny of 10 Oregon-R wild-type control females. Strains were classified according to the following criteria: 0–10% of wild-type progeny, sterile; 10–50%, semisterile; 50–75% subfertile.

Stocks

The generation of insertion strains and nomenclature is described by Bellen et al. (1989). The mutant strains used in this study are listed in Table 1.

Table 1.
graphic
graphic

General characteristics of β-galactosidase expression in ovaries of P[lArB] transposants

In order to explain the staining patterns that we observe in ovaries, a brief description of oogenesis in Drosophila is given below (see Fig. 1; for reviews, see King (1970), and Mahowald and Kambysellis, (1980)). Two fundamentally different cell lineages contribute to the formation of a mature Drosophila egg: cells derived from the germ line and cells derived from the soma. During oogenesis, each germ line pro-oocyte derived from a stem cell divides to produce an oocyte and 15 nurse cells whose cellular contents are later imported into the developing oocyte through intercellular cytoplasmic bridges. The somatic cells, named follicle cells, surround each developing egg and its sister nurse cells and secrete the protective coverings of the egg, such as the vitelline membrane and the chorion. The egg, nurse cells and surrounding monolayer of follicle cells together make up the egg chamber. A Drosophila ovary is composed of 10 to 20 ovarioles each containing egg chambers at a series of different developmental stages. The egg chambers at the earliest stages of oogenesis are located at the anterior tip of each ovariole, while more mature egg chambers are located more posteriorly. King (1970) has subdivided the development of the egg chamber into 14 stages on the basis of morphological characteristics (see Fig. 1).

Fig. 1.

(A) Drawing of two ovaries with two separated ovarioles. (B) Detail of the anterior end of an ovariole consisting of the germarium (g) and maturing egg chambers (tf; terminal filament; sc, stem cell; es, epithelial sheet; fc, follicle cells; fs, follicular stalk; ooc, oocyte: p, polar cells). Also shown are a stage 9 (C), a stage 10 (D) and a stage 13 (E) egg chamber (nc, nurse cells; be, border cells; rc, ring canal; sfe, squamous follicle cells; on, oocyte nucleus; efe, columnar follicle cells; da, dorsal appendages; mp, micropyle and ap, aeropyle). (A) to (E) after King, (1970). (F) Photograph of an ovary of transposant C55.1S3. The nurse cells and oocyte nucleus express β-galactosidase. The β-galactosidase expressed in the nurse cells is transported into the oocyte at the end of stage 10 and during stage 11.

Fig. 1.

(A) Drawing of two ovaries with two separated ovarioles. (B) Detail of the anterior end of an ovariole consisting of the germarium (g) and maturing egg chambers (tf; terminal filament; sc, stem cell; es, epithelial sheet; fc, follicle cells; fs, follicular stalk; ooc, oocyte: p, polar cells). Also shown are a stage 9 (C), a stage 10 (D) and a stage 13 (E) egg chamber (nc, nurse cells; be, border cells; rc, ring canal; sfe, squamous follicle cells; on, oocyte nucleus; efe, columnar follicle cells; da, dorsal appendages; mp, micropyle and ap, aeropyle). (A) to (E) after King, (1970). (F) Photograph of an ovary of transposant C55.1S3. The nurse cells and oocyte nucleus express β-galactosidase. The β-galactosidase expressed in the nurse cells is transported into the oocyte at the end of stage 10 and during stage 11.

Ovaries of wild-type females were stained using X-gal (5-bromo-4-chloro-3-indolyl β-D-galactoside) in order to determine whether any endogenous ovarian enzymes might react with the chromogenic substrate. Endogenous activity was detected at the posterior end of each ovariole at the junction with the lateral oviduct, and at the base of the dorsal appendages in stage 14 oocytes (see also Fasano and Kerridge, 1988). This staining appears diffuse and does not seem to be localized in a specific cellular compartment. This is in sharp contrast to the staining patterns observed in the ovaries of flies that carry the enhancer detector, P[lArB]. Analysis of ovarian whole mounts from these strains suggests that the P-transposase-β-galactosidase fusion protein encoded by P[lArB] is localized to the nuclei of cells in which it is present. Similar observations have been made in P[lArB] transposant embryos (Bellen et al. 1989). Internal nuclear localization was confirmed by staining ovarian sections (see Fig. 2). We conclude that the β-galactosidase fusion protein contains a nuclear targeting sequence which is presumably located in the 128 amino-terminal amino acids derived from the P-transposase protein.

Fig. 2.

(A) Section and (B) camera-lucida drawing of part of an adult female abdomen of a strain in which follicle cells and nurse cells express β-galactosidase activity. Staining with X-gal shows that the β-galactosidase activity is confined to the nuclei of nurse cells and follicle cells whereas endogenous β-galactosidase activity in the gut epithelium is located in the cytoplasm. Nuclear localization has been confirmed by double staining ovarian sections with X-gal and a nuclear specific dye (data not shown).

Fig. 2.

(A) Section and (B) camera-lucida drawing of part of an adult female abdomen of a strain in which follicle cells and nurse cells express β-galactosidase activity. Staining with X-gal shows that the β-galactosidase activity is confined to the nuclei of nurse cells and follicle cells whereas endogenous β-galactosidase activity in the gut epithelium is located in the cytoplasm. Nuclear localization has been confirmed by double staining ovarian sections with X-gal and a nuclear specific dye (data not shown).

Ovaries of females from a total of nearly 600 independent strains carrying single insertions of the novel P-element enhancer detector, P[lArB] (Bellen et al. 1989; Wilson et al. 1989), were stained for β-galactosidase activity. The insertions are located on the X-chromosome (81 strains), the CyO second chromosome (235 strains) and the ry506 or TM2 third chromosomes (270 strains). About 47% of all strains show β-galactosidase activity in one or more ovarian cells which do not stain in wild-type flies. The staining patterns in almost all of these transposants are temporally and spatially regulated during oogenesis. Fig. 3 A, B and C show egg chambers from three different transposants in which, respectively, germ line cells, follicle cells, and both germ line and follicle cells are stained. Table 2 gives an overall summary of the staining data. Approximately equal numbers of transposants are stained in germ line cells (19%), follicle cells (10%) or in both cell types (17%). This is generally in agreement with the distribution frequency reported by Fasano and Kerridge (1988). However, they found that 68%, as opposed to 47%, of all strains stained in the ovaries; the additional number of β-galactosidase-positive strains can all be accounted for in the class of insertion strains stained only in follicle cells (33 % as opposed to 10%). We have no obvious explanation for this discrepancy except, perhaps, the difference in sample size between the two experiments.

Table 2.

General distribution of staining patterns

General distribution of staining patterns
General distribution of staining patterns
Fig. 3.

Egg chambers of transposants expressing /3-galactosidase in ovarian cells. In all cases, anterior is to the left. The cells that stain and the stage of oogenesis are shown in brackets. (A) A 294.1F3 (nurse cells, oocyte nucleus; stage 10); (B) A333.2F2 (all follicle cells including the border cells; stage 10). (C) A186.1F1 (nurse cells, oocyte nucleus, columnar follicle cells; stage 10). (D) A464.1M3 (oocyte; stage 10); (E) A186.1F1 (nurse cells at anterior pole, some columnar follicle cells; stage 10). (F) B52.1M3 (columnar follicle cells; stage 10). (G) B2.1M2 (squamous follicle cells (arrowheads); stage 10).(H) A418.1M2 (follicle cells at anterior pole; stage 7).(I) A450.1F1 (follicle cells in the middle of the egg chamber; stage 8). (J) A208.1F3 (ring of follicle cells at the nurse cell/oocyte junction, ventral view; stage 10). (K) B38.2M2 (stem cells in germaria (arrowhead), follicle cells initially only at posterior pole and later evolving into a double gradient of stained cells; stages 1–10). (L) A175.1M3 (aeropyle; stage 10). (M) B36.1M3 (border cells; stage 10). (N) A374.1F3 (polar cells; stages 1–8). (O) B17.1M2 (polar cells among border cells, polar cells at posterior pole; stage 10). (P) and (Q) A534.3M3 (follicle cells first forming a belt and then a double gradient; stages 1–10 and stage 10). (R) B73.1M2 (nurse cells, follicle cells forming a double gradient; stage 10).

Fig. 3.

Egg chambers of transposants expressing /3-galactosidase in ovarian cells. In all cases, anterior is to the left. The cells that stain and the stage of oogenesis are shown in brackets. (A) A 294.1F3 (nurse cells, oocyte nucleus; stage 10); (B) A333.2F2 (all follicle cells including the border cells; stage 10). (C) A186.1F1 (nurse cells, oocyte nucleus, columnar follicle cells; stage 10). (D) A464.1M3 (oocyte; stage 10); (E) A186.1F1 (nurse cells at anterior pole, some columnar follicle cells; stage 10). (F) B52.1M3 (columnar follicle cells; stage 10). (G) B2.1M2 (squamous follicle cells (arrowheads); stage 10).(H) A418.1M2 (follicle cells at anterior pole; stage 7).(I) A450.1F1 (follicle cells in the middle of the egg chamber; stage 8). (J) A208.1F3 (ring of follicle cells at the nurse cell/oocyte junction, ventral view; stage 10). (K) B38.2M2 (stem cells in germaria (arrowhead), follicle cells initially only at posterior pole and later evolving into a double gradient of stained cells; stages 1–10). (L) A175.1M3 (aeropyle; stage 10). (M) B36.1M3 (border cells; stage 10). (N) A374.1F3 (polar cells; stages 1–8). (O) B17.1M2 (polar cells among border cells, polar cells at posterior pole; stage 10). (P) and (Q) A534.3M3 (follicle cells first forming a belt and then a double gradient; stages 1–10 and stage 10). (R) B73.1M2 (nurse cells, follicle cells forming a double gradient; stage 10).

Fasano and Kerridge (1988) also reported that lines expressing β-galactosidase in both germ line and follicle cells show an even distribution of β-galactosidase in all ovarian cells. We observe that this is indeed the case in some transposant strains, but we have also identified 53 insertion strains (9 % of the total number of transposants) that express β-galactosidase in the germ line and only in subsets of follicle cells (for example, see Fig. 3R) or in groups of follicle cells staining only at particular stages. Some of these strains will be discussed below.

Germ line-specific expression patterns

The anterior tip of each ovariole consists of the germarium (see Fig. 1), which contains two or three stem cells (Wieschaus and Szabad, 1979). These cells are derived from embryonic pole cells and give rise to all the more differentiated germ line cells. Each stem cell can divide, producing another stem cell and an oogonium. The oogonium subsequently divides four times to form a 16 cell cyst. These divisions are incomplete so that the sister cells are connected by cytoplasmic bridges called ring canals. Only two cells have the maximum number of four ring canals; one of these differentiates into an oocyte. The other 15 cells in the cyst form nurse cells. The nurse cells become polyploid, have very large nuclei and contribute most of their contents to the ooplasm as development proceeds.

Table 3 summarizes the expression patterns observed in germ line cells. Transposants that stain exclusively in the germ line cells can be subdivided into six subgroups as shown in Table 3. Most of these transposants express β-galactosidase only in the nurse cells. A single strain expressed β-galactosidase only in the oocyte nucleus: no β-galactosidase activity was observed in the nurse cells but the ooplasm stained strongly (see Fig. 3D; this strain has since been lost). Five transposants express β-galactosidase at different levels in different nurse cells. Some of these transposants exhibit highest activity in the most anterior nurse cells (Fig. 3E), whereas other transposants exhibit highest activity in the most posterior nurse cells (not shown). These results suggest that different nurse cells play different functional roles during oogenesis. Further studies will be required to investigate whether such differential gene regulation leads to a nonuniform localization of gene products in the ooplasm.

Table 3.

Distribution of expression patterns in the germ line

Distribution of expression patterns in the germ line
Distribution of expression patterns in the germ line

In many strains, the β-galactosidase expressed in the nurse cells is transported into the egg ooplasm at the end of stage 10 and during stage 11 (see Fig. 1F). Staining is then detected in the ooplasm but not in the oocyte nucleus. Most of these strains also stain for β-galactosidase activity in freshly laid eggs (Bellen et al. 1989). Some strains that express β-galactosidase weakly in nurse cells at stage 10 do not show staining later in the ooplasm. Both these transposants, and other strains that express β-galactosidase in nurse cells at early stages but not at later stages, are referred to as ‘transient expression’ strains.

The histogram shown in Fig. 4A summarizes the data on the temporal aspects of /3-galactosidase expression in the nurse cells. Most transposants that express β- galactosidase before stage 6 are stained at stage 1 and continue to be stained during subsequent stages. From stage 6 onwards, an increasing number of transposants express β-galactosidase, so that most of the strains that are stained in the germ line show some staining at stage 10. Since the β-galactosidase starts to be transported into the oocyte at the end of stage 10, results for later stages are not presented. The temporal expression patterns suggest that genes that are expressed in the nurse cells can be broadly subdivided into two classes; genes that are activated very early in oogenesis (about 15%), and genes that are only expressed during later stages, from stage 6 onwards and especially at stages 9 and 10 (about 85 %).

Fig. 4.

Temporal distribution of β-galactosidase activity in (A) the nurse cells, (B) the oocyte nucleus and (C) follicle cells of 587 transposants. Staining after stage 10 was not recorded for nurse cells and oocyte nuclei as the β-galactosidase produced in the nurse cells is transported into the oocyte at the end of stage 10 and during stage 11 (see text).

Fig. 4.

Temporal distribution of β-galactosidase activity in (A) the nurse cells, (B) the oocyte nucleus and (C) follicle cells of 587 transposants. Staining after stage 10 was not recorded for nurse cells and oocyte nuclei as the β-galactosidase produced in the nurse cells is transported into the oocyte at the end of stage 10 and during stage 11 (see text).

In order to interpret data on staining in the oocyte nucleus it is first necessary to outline a number of observations and assumptions that were made with respect to the β-galactosidase expression in the oocyte. Many of the strains that express β-galactosidase strongly in the nurse cells before stage 10 do not show staining in the oocyte nucleus. However, for a number of strains we observe that the oocyte nucleus also contains β-galactosidase (see Fig. 3A,C), suggesting that the β-galactosidase fusion gene is expressed in the oocyte as well as the nurse cells in these transposants. With this assumption, Fig. 4B summarizes the temporal expression patterns observed in the oocyte nucleus. It is more difficult to assess -galactosidase expression in the oocyte after stage 10, since by this time the enzyme produced in the nurse cells has been released into the ooplasm and may mask oocyte-specific staining. Strains that show no staining activity in the oocyte nucleus at stage 10, but do express β-galactosidase in the nurse cells, often show a different staining pattern in the oocyte nucleus at stage 11 and 12 when compared to strains that show staining in the nuclei of both nurse cells and the oocyte at stage 10. The former usually show no difference in staining intensity between the ooplasm and the oocyte nuclei, whereas the latter often show dark-staining oocyte nuclei in a fainter back ground. However, a number of transposants express β- galactosidase at such high levels in the nurse cells and later in the ooplasm that it is impossible to identify the oocyte nucleus after stage 10 in these strains (see for example Fig. 1F). We have therefore not presented data on β-galactosidase expression in the oocyte after stage 10.

One interpretation of our results might be that β- galactosidase synthesized in the oocyte is more effectively targeted to the oocyte nucleus than enzyme transported from the nurse cells. However, in the one strain that expressed β-galactosidase exclusively in the oocyte (from stage 10) and which could therefore be analyzed after stage 10, it is clear that much of the β-galactosidase is localized to the ooplasm (see Fig. 3D). Therefore, an alternative hypothesis, which might better explain our results, is that β-galactosidase present in the ooplasm before stage 10 is more efficiently targeted to the oocyte nucleus (or taken up by the nucleus) than at later stages.

As shown in Fig. 4 B, no staining was observed in the oocyte nucleus during the first five stages of oogenesis. In total, many fewer transposants express β-galactosidase in the oocyte nucleus than in the nurse cells. These observations are in agreement with previous findings by King and Burnett (1959) and Mahowald and Tiefert (1970). However, our data suggest that a significant number of genes may be active in the oocyte nucleus between stages 7 and 10, whereas previous studies using [3H]uridine detected RNA synthesis only in stage 10 oocyte nuclei (Mahowald and Tiefert, 1970). One obvious explanation for this discrepancy is that enhancer detection may be more sensitive than simple autoradiographic analysis. It should be emphasized that we found only one transposant that specifically labeled the oocyte nucleus and the ooplasm. The only gene that is known to be expressed specifically in the oocyte is the fs(l)K10 gene (Haenlin et al. 1987). Based on these observations, we propose that very few genes are expressed specifically in the oocyte nucleus, while many more genes may be expressed in both the oocyte nucleus and other ovarian cells.

Somatic cell-specific expression patterns

Approximately 80 follicle cells surround the germ line cells in stage 1 egg chambers. These follicle cells divide during subsequent stages to give rise to approximately 1200 cells per egg chamber. About 90 % of the follicle cells will eventually form a high columnar epithelial layer around the oocyte (see Fig. 1). These cells secrete yolk into the ooplasm and produce the vitelline membrane and the chorion of the oocyte. Most of the remaining 10% of cells surround the nurse cells and form a thin squamous epithelium. About 6 to 10 follicle cells located at the most anterior end of the egg chamber differentiate into border cells at about stage 8. These cells migrate between the nurse cells towards the anterior end of the oocyte where they contribute to the formation of the micropyle, the entry point for the sperm. Another set of follicle cells located peripherally and bordering the oocyte-nurse cell junction migrate into the egg chamber on the anterior side of the oocyte at stage 10. These cells will contribute to the formation of the dorsal appendages and the operculum, the exit point of the first instar larva. Finally, some follicle cells at the posterior end of the egg chamber will form the aeropyle, a structure that is thought to be involved in respiration (Margaritis et al. 1980).

When the PflArB] transposant ovaries were analyzed, a great variety of staining patterns was observed in follicle cells (see also Fasano and Kerridge, 1988). Some of these patterns are shown in Figs 3 F to Q and the staining data are summarized in Table 4. Most transposants show β-galactosidase expression in subpopulations of follicle cells that have previously been recognized on the basis of their morphological characteristics (Margaritis et al. 1980). Like Fasano and Kerridge (1988), we also found strains in which subsets of follicle cells are stained that have not been recognized as morphologically different from their neighbours. For example, the transposant shown in Fig. 31 expresses β- galactosidase in a belt of follicle cells located around the middle of the oocyte.

Table 4.

Distribution of expression patterns in follicle cells

Distribution of expression patterns in follicle cells
Distribution of expression patterns in follicle cells

In addition, we have noted that several transposants show a graded expression of β-galactosidase in the follicle cells. Staining may be highest in the most anterior follicle cells and decrease in more posteriorly located cells (Fig. 3H). Alternatively, the most posterior follicle cells may express the highest level of enzyme, which then decreases in more anteriorly located cells (Fig. 3K). A third type of gradient was observed in three strains in which the strongest expression is found in the follicle cells overlying the anterodorsal surface of the oocyte; β-galactosidase activity decreases in more ventral and more posterior follicle cells (Fig. 3 P-R). We refer to this pattern as the ‘double gradient’ pattern. In one transposant, expression in the follicle cells is first observed in a belt at stage 6 and the double gradient is only established during the subsequent stages (see Fig. 3P, Q). Another of these transposants also expresses β-galactosidase in the nurse cells (Fig. 3R). To our knowledge, there are presently no reports of genes that show such transcript or protein distributions.

Recently it has been shown that follicle cells play a role in specifying the dorsoventral polarity of the egg chamber. Two fs loci, gurken and torpedo, cause a ventralization of the follicle cell epithelium and a similar ventralization of the embryo (Schiipbach, 1987). The graded follicular expression patterns may therefore reflect the expression pattern of a gene that is either involved in the establishment of anterior-posterior and dorsal-ventral polarities in the egg, or is regulated by genes that establish these polarities. Two insertions in transposants that show such gradients of β-galactosidase activity in follicle cells have been cytologically mapped (see Table 5). Although several mutations map near or at the cytological positions of these insertions, none causes a maternal effect on polarity in the embryo. These transposants are presently being studied more extensively.

Table 5.

Cytological mapping positions*

Cytological mapping positions*
Cytological mapping positions*

Although, in many transposants (67% or 107 of 159 strains that are stained in the follicle cells at stages 9 and 10), staining is observed in the major follicular cell types (either the squamous or columnar follicle cells), a significant number of transposants express β-galactosidase in more specialized ovarian somatic cells. In particular, a large proportion of transposants express β- galactosidase in the border cells or in the follicle cells at the nurse cell-oocyte junction; these cell types have complex migration pathways at stages 9 and 10. We observe that 28% (45/159) of transposants show staining in the border cells and 14% (23/159) in the follicle cells around the nurse cell-oocyte junction. Other follicle cells, which do not show a complex migration pattern, such as the most posterior follicle cells of the egg chamber, stain in many less transposants (6/159) at stages 9 and 10. The high number of transposants that express β-galactosidase in migrating follicle cells may reflect the complex requirements of these cells for migration and differentiation.

In general, staining patterns in the ovaries of transposants are quite complex, and only a few strains express β-galactosidase solely in a very specific subpopulation of follicle cells. For example, two strains stain only the border cells (see Fig. 3M) whereas 43 strains were identified in which border cells stained in combination with other ovarian cells, including the nurse cells in some cases. Similarly, 9 transposants stain only in follicle cells at the oocyte-nurse cell junction (Fig. 3J), whereas 14 further transposants express β-galactosidase in these cells and other ovarian cells.

The complexity of some patterns also varies considerably according to the developmental stage of the egg chamber. For example, strains that are stained in the two pairs of polar cells, one pair of which is located at the anterior end of the egg chamber and the other at the posterior end, often show simple staining patterns at early stages (see Fig. 3N) and more complex patterns during later stages. In all transposants, the anterior pair of cells comigrates with the border cells (see Fig. 30). The posterior pair, on the other hand, is often found among a cap of stained cells at the posterior end of the egg chamber during later stages (not shown). The function of the polar cells is unknown but it has been proposed that they may be involved in the overall control of the polarity of the developing egg chamber (Brower et al. 1981), and may therefore have some functional similarity to the ‘distal tip cells’ in the gonads of the nematode Caenorhabditis elegans (Kimble and White, 1981).

The temporal distribution of β-galactosidase expression in transposants that are stained in follicle cells is summarized in the histogram shown in Fig. 4C. There appear to be two major phases when β-galactosidase expression is first detected; the first occurs at the earliest stages of oogenesis while the second begins at stage 5. Roughly 50% (77/148) of all strains first express β-galactosidase at stage 9 and 10. The similarity between the temporal distributions for follicle cell and nurse cell β-galactosidase expression (compare Fig. 4A, C) may reflect the close coordination of development in these two cell types that is probably required during oogenesis (for discussion of possible cell-cell interactions, see Parks and Spradling, 1987).

Analysis of P[lArB]-associated mutants

Transposant strains carrying insertions on the X-chromosome or third chromosome were analysed to test whether the insertion was associated with a recessive mutant phenotype. Since the CyO second chromosome carries a lethal mutation, it was not possible to analyze the second chromosome insertions in this way. Five transposants were identified that have decreased fertility and a further three were initially recognized on the basis of an aberrant ovarian phenotype. In order to establish that the P-element insertion is the cause of the mutant phenotype in these transposants, we generated flies in which the P[lArB] insertion had been excised by mating the transposants to flies carrying a genomic source of P-transposase (Robertson et al. 1988; see Materials and methods). For seven of the eight transposants, some of the resulting chromosomes no longer carried the mutation, suggesting that the observed phenotype is linked to the P[lArB] insertion. The characteristics of the mutant strains are summarized in Table 6 and some of the defective ovaries and egg chambers are shown in Fig. 5. Two mutant strains show no ovarian defects. Most of the other mutations exhibit incomplete penetrance of the visible ovarian defects. Three mutants have egg chambers with too many or too few nurse cells. Ovaries of the A309.1M3 females are highly aberrant and most egg chambers are tumorous; some have nurse cell nuclei on both sides of the oocyte (see Fig. 5H, I).

Table 6.

Mutations affecting oogenesis*

Mutations affecting oogenesis*
Mutations affecting oogenesis*
Fig. 5.

Ovarian staining patterns of transposants in which insertion of P[lArB] causes a mutation leading to ovarian defects. The stained cells in heterozygous flies and the stage of oogenesis of the egg chambers in the figure are shown in brackets. (A) strain A275.2F1 - the arrowhead points towards an egg chamber with inversed polarity (stages 1 to 10) and (B) A275.2F1 - enlargement of (A), arrowheads mark nurse cell nuclei within the ooplasm in another egg chamber (nurse cells, oocyte nucleus, polar cells, squamous and columnar follicle cells; stage 10). (C) 285.2F1 - a defective ovary and (D) A285.2F1 - enlargement of three egg chambers (nurse cells, oocyte nucleus; stages 7, 9 and 10). (E) A467.1F1 - egg chamber with too many nurse cells (stage 10) and (F) A467.1F1 - the arrowhead marks two nuclei in the ooplasm (nurse ceils, oocyte nucleus, follicle cells (faint); stage 10). (G) A33.1M3 - the four arrowheads mark the region where the nurse cells have not disintegrated properly (nurse cells, oocyte nucleus; stages 9 and 10). (H) A309.1M3 - a tumorous egg chamber is shown (stage 10) and (I) A309.1M3 - nurse cells on both sides of the oocyte, three nuclei in the ooplasm (nurse cells; stage 10).

Fig. 5.

Ovarian staining patterns of transposants in which insertion of P[lArB] causes a mutation leading to ovarian defects. The stained cells in heterozygous flies and the stage of oogenesis of the egg chambers in the figure are shown in brackets. (A) strain A275.2F1 - the arrowhead points towards an egg chamber with inversed polarity (stages 1 to 10) and (B) A275.2F1 - enlargement of (A), arrowheads mark nurse cell nuclei within the ooplasm in another egg chamber (nurse cells, oocyte nucleus, polar cells, squamous and columnar follicle cells; stage 10). (C) 285.2F1 - a defective ovary and (D) A285.2F1 - enlargement of three egg chambers (nurse cells, oocyte nucleus; stages 7, 9 and 10). (E) A467.1F1 - egg chamber with too many nurse cells (stage 10) and (F) A467.1F1 - the arrowhead marks two nuclei in the ooplasm (nurse ceils, oocyte nucleus, follicle cells (faint); stage 10). (G) A33.1M3 - the four arrowheads mark the region where the nurse cells have not disintegrated properly (nurse cells, oocyte nucleus; stages 9 and 10). (H) A309.1M3 - a tumorous egg chamber is shown (stage 10) and (I) A309.1M3 - nurse cells on both sides of the oocyte, three nuclei in the ooplasm (nurse cells; stage 10).

We have focused our attention on females of strain A33.1M3 because they produce small eggs with short, abnormally positioned dorsal appendages and are essentially sterile (7 % of the progeny produced by wildtype controls), a phenotype which shows some resemblance with previously isolated fs mutations (see Schüp-bach, 1987). We have observed that the nurse cells do not regress completely between the dorsal appendages in egg chambers of the A33.1M3 females as is normally observed in wild-type strains (see Fig. 5G). In experiments to revert the mutant phenotype by excision, 17 strains were recovered that had lost the rosy+ eye color marker of P[lArB]: two exhibit a significantly increased fertility when compared to homozygous A33.1M3 females (40 % to 60 % of the progeny from the wild-type control) and do not produce morphologically abnormal eggs or display the mutant ovarian phenotype. The other 15 strains lacking the rosy+ marker all show a more extreme recessive mutant phenotype compared to A33.1M3. Two of these strains are completely sterile and lay no eggs. They have defective ovaries in which egg chambers that have developed post stage 6 are morphologically abnormal. Heterozygous females containing a copy of either of these female sterile chromosomes and the original A33.1M3 chromosome produce eggs with the same phenotype as homozygous A33.1M3 females. The other 13 strains recovered are homozygous lethal. Heterozygous females containing a copy of either of the female sterile rosy+ chromosomes and one of the lethal chromosomes are sterile (less than 5 % of the progeny produced by wild-type controls). In the light of these observations and the recovery of partial revenants, we conclude that P[lArB] is probably responsible for the mutant phenotype and that the strains we have generated carry a mutation in the same fs gene. Since noncomplementing lethal mutations are also recovered at high frequency, it is possible that the A33.1M3 mutant and the two other sterile mutants are hypomorphic alleles of an essential gene. This hypothesis is strengthened by the observation that complementation tests between A33.1M3 and previously isolated female sterile and maternal effect mutations mapping at or near the cytological mapping position of the P[lArB] insertion (83B) have not revealed any allelism with known genetic loci (see Table 6).

Wilson et al. (1989) and Bellen et al. (1989) have shown that, in a significant proportion of insertion strains with an embryonic staining pattern, a gene with a similar expression pattern lies near to the insert. In this report, we have assumed that this also applies to ovarian staining patterns. One prediction that may be made from this assumption is that most of the mutant transposant strains that are partially sterile or have a mutant ovarian phenotype will be stained in ovaries. Indeed, all seven strains express β-galactosidase in ovaries. More remarkably, all seven strains are stained in nurse cells either at or before stage 9. We have shown that only 14% of all transposant strains express β- galactosidase in nurse cells at these early stages, suggesting that there is a considerable bias for selecting such staining patterns in P[lArB]-associated mutants. Of course, since three of the strains were first identified on the basis of their aberrant ovarian phenotype, staining in the nurse cells of these strains may have increased the likelihood of recognizing such a phenotype. However, even if only the four P[lArB]-associated mutants identified on the basis of sterility are considered, the probability that all four strains, if selected at random, would stain in the early nurse cells is about 0·05 %. We therefore conclude that there is a significant positive correlation between a mutant phenotype affecting oogenesis and a specific β-galactosidase expression pattern, although early nurse cell staining will almost certainly not be the only pattern associated with such mutants.

Detection of ovarian gene expression patterns by enhancer detection and applications of enhancer detection to the study of female sterile genes

Previous results with the enhancer detector technique have suggested that this method is an indicator of general gene expression patterns in Drosophila (Wilson et al. 1989; Bellen et al. 1989). The fact that P[lArB]-associated fs and ovarian mutants in our screen are stained in the ovaries is at least consistent with this hypothesis. Nevertheless, it has been suggested that P-elements may insert preferentially into chromosomal regions that are actively transcribed (for review see Engels, 1988). Therefore, it is possible that there is a bias towards insertion near genes that are expressed in the male germ line since that is where transposition occurs. However, comparison of our data with estimates made from genetic analyses does not reveal any obvious bias towards genes expressed in the female germ line. For example, based on genetic evidence it has been shown that 50 % (Perrimon and Gans, 1983) to 75 % (Perrimon et al. 1986) of the tested recessive female sterile mutations and about 70 % of the zygotic lethal mutations on the X-chromosome (Perrimon et al. 1989) affect genes that are expressed in the germ line. In comparison, our data suggests that roughly 76% (211/ (587–312)) of all genes expressed in ovarian cells are transcribed in the nurse cells or germarium. Furthermore, Perrimon et al. (1986) have proposed that very few genes are exclusively expressed during oogenesis. Although we have not analysed the transposants at all stages of development, we have stained embryos (Bellen et al. 1989), imaginai discs and brain of third instar larvae (Greg Gibson, personal communication), as well as ovaries in all the strains tested. Only 2/587 transposants stain exclusively in ovaries. Hence, our observations substantiate the conclusions and data of Perrimon et al. (1986), and suggest that less than 1 % of all genes may be expressed only in ovaries.

Based on previous genetic screens for fs mutants and some statistical assumptions, it has been estimated that approximately 10–15 % of genes in the Drosophila genome are required for oogenesis (Gans et al. 1975; Mohler, 1977; King and Mohler, 1975). However, Perrimon et al. (1984 and 1986) have presented evidence that a substantially larger fraction of Drosophila genes is needed for fertility. Our results provide an estimate of roughly 50 % for the proportion of genes expressed in the ovary, even if their expression is not absolutely required during oogenesis.

The enhancer detection method presumably identifies a more extensive group of genes expressed in the ovary than genetic screens for fs genes. Hence, in the light of previous results (Bellen et al. 1989; Wilson et al. 1989) and those presented here, it is possible to suggest at least two approaches for the cloning and genetic identification of important ovarian genes using enhancer detectors. The first involves the characterization of genes associated with specific /3-galactosidase staining patterns. For example, we are studying a small number of transposants that stain in only specific columnar follicular cells surrounding the oocyte (e.g. the follicular pattern gradients). Genes controlled by the detected regulatory elements may have some role in initially determining or maintaining the polarity of the egg chamber. The P[lArB] insertions in transposants that show interesting β-galactosidase staining-patterns can easily be cytologically mapped (see Table 5 and 6). An important advantage of the enhancer detector technique is that a genetic study of the region may be easily undertaken (Bellen et al. 1989). Particularly as P-elements often insert in the proximity of the promoter of a gene (Engels, 1988; Wilson et al. 1989), it may be possible to recover mutant alleles of the gene of interest by imprecise excision of PflArB], The available duplications, deficiencies or point mutations that respectively cover, uncover or map at the cytological interval of the P[lArB] insertion can then be tested in complementation assays with the fs or lethal mutations that are recovered by imprecise excision of the P-element. Such experiments may allow the function of the newly identified gene to be determined.

A second approach is to use enhancer detector transposons as mutagens; the resulting staining patterns associated with these mutants may then be analyzed and appropriate transposants selected. This should be particularly useful in the study of lethal mutations since only those mutant transposants that stain in the ovary might be selected for further investigation. Furthermore, our evidence suggests that certain specific expression patterns, such as early nurse cell staining, are more frequently associated with insertions near genes required for oogenesis than other patterns. Thus, even in a large enhancer detector screen for lethal mutations, it should be possible on the basis of staining to select a smaller number of strains with mutations in genes that are not only essential but also likely to be involved in oogenesis.

We would like to thank Cahir O’Kane and Rebecca Kurth Pearson for their help and advice throughout the course of this work. Our thanks also go to Christiane Niisslein-Volhard, Norbert Perrimon, Madeleine Gans, Dawson Mohler, Robert King, David Glover and the Umea stock center for mutant fly stocks. We are grateful to Greg Gibson for information supplied before publication and for helpful comments on the manuscript. Final thanks go to Erika Wenger-Marquardt for efficiently typing the manuscript. This work was supported by a fellowship of the Kanton Bern to U.G., a NATO fellowship to H.B., a Royal Society exchange fellowship to C.W., the Swiss National Science Foundation, and the Kantons of Basel Stadt and Basel Landschaft.

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