MOLECULAR REPRODUCTION AND DEVELOPMENT 28:307-317 (1991)

Review Article Genetic and Molecular Analvsis of Maternal Information in Region 32 of Drosophila melarugaster CARLA MALVA, FRANC0 GRAZIANI, VALERIA CAVALIERE, ANDREA MANZI, AND ANGELA TIN0 International Institute of Genetics and Biophysics, CNR, Naples, Italy

INTRODUCTION Genetic analysis has been proved to be a very powerful tool in elucidating the molecular mechanisms involved in eukaryotic development. Pioneer studies allowing the identification of several Drosophila melanogaster developmental genes, later observed to be highly similar to vertebrate genes, have shown that the work carried out on this insect plays an important role also in the understanding of developmental processes in higher eukaryotes, including humans. Early embryonic development depends on complex processes occurring during oogenesis which, in D. melanogaster, can easily be approached on the genetic, cellular and molecular levels. Different cell types of different origins are involved in Drosophila oogenesis (King, 1970; Mahowald and Kambysellis, 1980; Konrad et al., 1985). Germ cells segregate early in the posterior part of the developing embryo in a small group of polar cells (Mahowald, 1962; Foe and Alberts, 19831, which, during gastrulation, are carried into the embryo within the posterior midgut invagination. In embryos and larvae, germ cells behave as a closed system, cells growing logarithmically and both sister cells remaining after each division in the germ cell population (Illmensee et al., 1976).Somatic cell cytodifferentiation of the ovary begins during the third instar, and that of germ cells begins after the puparium formation with the establishment of an anterior population of stem cells, three for each ovarioles (about 15),which form the two ovaries of Drosophila. The stem cells divide asymmetrically so that one continues as stem cell and the other becomes a cystoblast. Each cystoblast undergoes four incomplete divisions forming the 16-cell cluster surrounded by follicle cells of mesodermal origin. The oocyte is, therefore, a member of the 16-sister-cell cluster, the other 15 functioning as nurse cells. The continuous process of oogenesis is divided into 14 morphological stages (King, 1970) (Fig. 1). That maternal genome is important in ensuring the correct embryonic development has been clear since the early observations that egg cytoplasm greatly contrib-

0 1991 WILEY-LISS, INC.

utes to the developing zygote. To study the expression of maternal genes involved in oogenesis and early embryogenesis, genetic analysis must naturally start with a collection of mutants with a vast variety of phenotypes sharing a common characteristic: The females are unable to generate offspring. The mutants can be grossly divided into two classes: 1) females unable to lay eggs or laying eggs with great morphological abnormalities (in this group are most of the gonadal- or female-sterility mutants and 2) females laying eggs without gross alterations but unable t o bear a correct embryonic development (in this group are most of the maternal-effect mutants). After considerable efforts of many groups, a large collection of mutants has been obtained. Thus the analysis of their different phenotypic aspects has enabled to identify several specific aspects of oogenesis or early embryogenesis for which the product of the wild-type gene is necessary (Lindsley and Grell, 1968; Zhalokar et al., 1975; Mohler, 197'7; Nusslein-Volhard, 1977; Nusslein-Volhard and Wieschaus, 1980; Komitopoulou et al., 1983; Perrimon et al., 1986; Orr et al., 1989; Schupbach and Wieschaus, 1989). Notwithstanding the highly numerous mutants identified until recently, it was difficult to assign a functional role to the various genes. It was therefore necessary to develop methods that would allow the identification of cells affected by the mutations. Two methods have become available, one consisting of pole cell transplantation (Illmensee, 1973; Van Deusen, 1977), the other making use of mitotic recombination (Wieschaus et al., 1978, 1981, Perrimon and Gans, 1983) to generate homozygous clones in ovaries carrying a dominant female sterile mutation (Dfs), now available for the major chromosomes (Perrimon, 1984; Erdelyi and Szabad, 1989). Both methods induce the

Received June 14, 1990; accepted September 19, 1990. Address reprint requests to Carla Malva, International Institute of Genetics and Biophysics, CNR, Via Marconi 10, 80125 Naples, Italy.

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Fig. 1. A dorsal view ofthe internal reproductive system of an adult Drosophila female, a diagram of a single ovariole and drawing of stages 8, IOB, 12 and 14.[Reproduced with permission from Konrad et al. (1985)Developmental Biology, Vol 1. Oogenesis: 577-617, Plenum Press, NY as modified from King, 1970.1

generation of genetic mosaics with a mutant germ line in a wild-type soma: When such a combination leads t o a mutant phenotype, then the mutation is germ line dependent, whereas, when the eggs produced are normal, then the gene is required only in somatic tissues. These two methodologies are nevertheless complex and present difficulties and limitations. Moreover, they can discriminate between germ line and soma but cannot indicate the type of cells affected. Finally, classical genetic approaches are unable to detect all the genes necessary for the production of functional eggs. In fact, it has been demonstrated that most of these genes are also necessary in other developmental stages and in tissues different from the ovaries: consequently, mutations in such genes will be zygotic lethal, and it will be impossible to assess the female sterility or the maternal effect produced by these genes. Useful in these cases can be the hypomorphic alleles, which sometimes allow adult survival but induce sterility because of the high amount of gene product required during oogenesis; however, they can be complicated to study because of the lack of clear-cut phenotypes. Recently, a very elegant method has been developed that allows one to identify in situ control elements of genes regulated during different developmental stages (O’Kaneand Gehring, 1987; Wilson et al., 1989; Bellen et al., 1989), including oogenesis (Grossniklaus et al., 1989): “P-element-mediated enhancer detection.” This transposon detects neighboring genomic transcriptional regulatory sequences by means of a P-galactosidase reporter gene, which, in the majority of cases, responds to nearby transcriptional regulatory sequences in the D . melanogaster genome. This method also allows evaluation of the number of genes involved in oogenesis. From the data of Grossniklaus et al. (19891, it is evident that about 50% of D. melanogaster genes are transcribed in the ovaries, but less than 1% are transcribed exclusively in the ovaries. These approaches provide information on the number and type of genes involved in oogenesis. When studying individual mutations, genetic analysis, together with the molecular approach and examination of the cellular phenotype, provides detailed knowledge of the role played by each single gene and the relationships between them. In this case, other more direct methodologies can be applied, such as in situ hybridization (Hafen et al., 1983; Tautz and Pfeifle, 1989) or DAPI staining (Lin et al., 1977; Coleman et al., 1981; Bansel and Pferiffer, 1985) of ovaries, isolated egg chambers and early embryos. Together these approaches have provided significant insight into the different stages of ovarian development and eggshell formation, the genesis of embryonic determination in egg cytoplasm, and the macromolecular functions involved in early embryonic development. Some of the most exciting results have come from the analysis of maternal genes whose products are involved in the establishment of the anteroposterior and dorsoventral polarity of the early embryo. The first mu-

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Fig. 2. A mapping of the mutants isolated by Sandler in 32 region of the left arm of chromosome 2 of Drosophila. [Reproduced with permission from Sandler, (1977) Genetics 86:56'7-582, Genetics Society of America, Chapel Hill, North Carolina.]

tants were isolated long ago (Nusslein-Volhard, 1977; Nusslein-Volhard and Wieschaus, 19801, and, after a great deal of time-consuming work to isolate and characterize the genes, the combination of all the available methodologies entered a particularly exciting phase in which the molecular hierarchies among these genes can be established and molecular models explaining the establishment of the coordinate axis and embryonic segments can be proposed (Nusslein-Volhard et al., 1987; Schupbach and Wieschaus, 1986; Schupbach, 1987;Tautz et al., 1987; Roth et al., 1989; Struhl et al., 1989; for a review see Ingham, 1988). Genetic and molecular analyses of female sterility mutants have been very useful in the dissection of the different phases of oogenesis such as cell division and differentiation (King, 1970), vitellogenesis (Barnet et al., 1980; Brennan et al., 19821, and eggshell formation (Komitopoulou et al., 1983; Orr et al., 19891,providing interesting information on the different roles played by germ cells and somatic tissues of ovaries. Among these two large groups of mutants, there occurs a small third group, which, interestingly, affects both the egg and embryo symmetry contemporaneously. The study of these mutants (Wieschaus et al., 1978; Schupbach, 1987) has allowed the hypothesis that follicular cells participate in regulating the deposition and distribution of positional signals in the oocyte for the establishment of both oocyte and embryonic polarity.

MATERNAL-EFFECT GENES IN REGION 32 In this general area is the work of our groups on four maternal effect genes hold up (hup),wavoid-like (wdl), daughterless-abo-like (dal),and abnormal oocyte (abo), located in region 32 of the standard salivary gland chromosome map in the euchromatic region of the left arm of D. mehogaster chromosome 2 (Sandler, 1977). The mutants are all recessive, hypomorphic, maternal effect, and female semisterile. Of these four mutants, abo is the only one that originates in the wild (isolated by Sandler in 1965 on the outskirts of Rome);the others are EMS-induced mutants isolated by Sandler (1977) while attempting to identify other abo alleles (Fig. 2).

The maternal effect shared by all these mutations, together with their very close association, seems to imply that their coordinated expression and function are necessary during oogenesis.

The abnormal oocyte Phenotype The abnormal oocyte mutation confers no visible phenotype on homozygous abo males or females, but homozygous abo females produce defective eggs. The probability of their developing into adults is much lower than that of heterozygous sister females and can be influenced by particular heterochromatic regions in the mother and/or in the zygote (Sandler, 1970, 1975, 1977; Parry and Sandler, 1974; Pimpinelli et al., 1985; Malva et al., 1985). Because of this interaction with heterochromatin, abolabo females carryingwild-type X chromosomes, when crossed to attached XY/O; abo+/ abo+ males, produce an excessive number of female offspring as a result of increased X/O embryonic lethality compared with X/XY zygotes produced (Sandler, 1970). An intriguing feature of the abo mutation consists of the loss of the abo phenotype. It is reported (Krider and Levine, 1975) that in homozygous abo stocks the abo maternal effect gradually decreasexas measured by the sex ratio in crosses to attached XY/O males, and is lost after several generations in homozygous conditions (aboGn). Krider and Levine (1975) had also observed the recovery of the abo mutation from strains that had lost the abo phenotype. The obvious explanation was that the loss of the abo phenotype is not due t o a selection of revertant abo+ flies. It had been thought that the heterochromatic segments interacting with abo influence in turn the structural cistrons for ribosomal RNA located within the sex heterochromatin (Ritossa et al., 1966) because, during loss of the abo phenotype, the rDNA content of each X chromosome increases (Krider and Levine, 1975; Krider et al., 1979; Yedvobnick et al., 1980). Having long been interested in the regulation of rDNA redundancy, we started our work on the abnormal oocyte mutant and demonstrated that the rDNA increase is associated with variations in

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Fig. 3. Southern blot analysis using the single recombinant phages digested with EcoRl and hybridized with 18 hr embryo (A)- and female (B)-labeledcDNAs. As a control, a n increasing amount of actin gene was used on top of the gels a s a marker of the specific activity of

the cDNA preparations (indicated with arrows). A scheme of the chromosome walking is also indicated. The EcoRl sites on the phages are those obtained to perform the walk. A more precise map was obtained for the single phages as the analysis proceeded.

wdl, and dal mutants are very poorly characterized, and their role during Drosophila development is still unknown. All the experiments carried out on these mutants are reported by Sandler (19771, with these conclusions: “1)all are recessive, hypomorphic mutations as evidenced by lethality or semi-lethality when heterozygous with a deficiency, survival in homozygous conditions and wild-type behavior when heterozygous The hup, wdl, and dal Mutations with a normal allele; 2) none has any paternal effect; 3) Although many phenotypic aspects conferred by the all show sex-differential mortality as evidenced by abo mutation have been described in detail, the hup, unequal recovery of XX females and XY males from

the restriction pattern of the nontranscribed spacer (Graziani et al., 1981). Later, Manzi et al. (1986) reported that loss of the abo phenotype can also occur without an rDNA increase; therefore, we hypothesized that this phenomenon was due to other events occurring in other regions, possibly the heterochromatic regions.

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Fig. 4. Restriction map of the blood transposon identified in the abo stock and inserted in the wild-type region identified by the pA2 clone of phage E3 in our study of region 32.

homozygous mothers; 4) all produce a maternal effect resulting in zygotic mortality before egg hatch; 5) the consequence of the maternal effect, in each case, is affected by the X or Y chromosome heterocromatic content of zygotes developing from eggs produced by mutant mothers; 6) the consequence of the maternal effect is, in all cases, less severe at 19°C than it is at the normal rearing temperature of 25°C.” With respect to point 5 , the three mutants exhibit a set of properties suggesting that, like abo, they also interact with sex chromosome heterochromatin. In the case of hup, the Y chromosome interacts lethally with it; in the case of wdl, Xh interacts lethally with the maternal effect, whereas the Y chromosome improves it; regarding dal, both Xh and Y partially compensate the maternal effect, as in the case of abo. There is as yet no information on these functional interactions. The difficulties of working with semisterile mutants are stressed by Sandler in his 1977 paper: “Unfortunately, hup and wdl homozygous females are too sterile to permit performing the required experiments.” Consequently, certain aspects of the phenotype, such as the differential progeny recoveries observed with different doses of Xh and Y or the influence of a wild-type allele on the probability of that zygote surviving the maternal effect, were examined by Sandler only in the case of dal. The basis of the increased male recovery is ascribed to the reduced survival of XO males (0.21) compared with XY males (0.41);the surviving dal maternally induced abnormality in the egg is very much reduced in the absence of a wild-type allele of the gene. We have started a detailed genetic analysis in an

attempt to define several phenotypic aspects of these mutants: maternal effect, zygotic effect, paternal effect, temperature sensitivity, stage of lethality of the developing embryos, etc. Considering the amount of DNA contained in region 32, the potential genetic information had to be much vaster than that corresponding to the few mutants isolated in it. Hence, besides a more detailed genetic analysis, we decided to adopt a molecular approach in order to obtain new insight into the functions present in the region and to enable elucidation of the molecular mechanisms involved in the loss of the abo phenotype.

The Molecular Analysis Starting with plasmid clone 153B9 (kindly provided by Walter Gehring), containing a D. melanogaster segment from region 32D, we screened a Drosophila library constructed in the EMBL4 lambda phage vector with embryonic DNA from Oregon R stock and walked about 200 kb in this region, from band 32D to band 32E-F. We used the recombinant phages we had isolated to possibly correlate them with the mutations we were studying by the following approaches: 1)analysis of the transcription pattern of region 32 in wild-type and in hup, wdl, dal, and abo stocks during the different developmental stages by Northern blots and screening of stage-specific cDNA libraries and 2) investigation of the molecular organization of region 32 to identify possible restriction enzyme site polymorphisms caused by the presence of one of our mutations. To acquire a general view of the gene expression in region 32, we hybridized restriction fragments of the

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recombinant phages isolated during the chromosome walk in that region with labeled cDNA prepared from poly-A’ RNAs extracted from different developmental stages of Drosophila flies (embryos, larvae, pupae, adult females, and adult males). We discovered interesting features and identified genes selectively expressed in particular developmental stages. Figure 3 shows the results obtained with 18 hr embryo (Fig. 3A) and female (Fig. 3B)-labeled cDNAs, hybridized on EcoR1-digested phages. As a control, an increasing amount of actin gene was used on top of the gels as a marker of the specific activity of the cDNA preparations (indicated with arrows in Fig. 3). We called the overlapping phages “D” and “E,” referring to the polytene bands of region 32 on which they hybridize, using progressive numbers to indicate the overlapping phages that hybridize on the same band. Obviously, the signals identified with this approach represent an underestimation of the transcripts present in the region due either to eventual truncations in the 5’ direction during cDNA synthesis or t o a very small amount of transcript, which we later verified when we studied in detail each single fragment of each single phage.

The 32E Vitelline Membrane Protein Gene As can be seen in Figure 3, phage E2 has an EcoRl fragment strongly hybridizing with a transcript present in females and not in 18 hr embryos, as is the case with a maternal transcript. We started our characterization with this fragment: the identified gene has an ovary-specific expression and, we discovered, codes for one of the vitelline membrane proteins (VMP) (Gigliotti et al., 1986; Graziani et al., 1986). The latter was an exciting result because a t that time only a partial cDNA (Mindrinos et al., 1985) had been described as corresponding to a VMP gene. Even now, only a few genes have been isolated and characterized in detail at positions 26A, 34C, and 32E (Burke et al., 1987; Popodi et al., 1988; Scherer et al., 1988; Gigliotti et al., 19891,although at least ten classes of VMP have been identified (King and Mohler, 1975; Petri et al., 1976; Fargnoli and Waring, 1982). The VMP gene we isolated from region 32E (VMP32E) was characterized in detail. A peculiar feature of the VMP gene family was discovered: a conserved hydrophobic domain, constituted of about 30 amino acids, is present in all the genes (Popodi et al., 1988; Scherer et al., 1988; Gigliotti et al., 1989). Synthesis of the proteinaceous eggshell in D.melanogaster is a system particularly well suited for cis-regulatory signals controlling gene expression in eukaryotes (Komitopoulou et al., 1983; Mariani et al., 1987; Mitsialis et al., 1987; Parks and Spradling, 1987; Orr et al.? 1989; Tolias and Kafatos, 1990). In a tissue-specific and temporally regulated fashion, several structural proteins are produced in rapid succession and according to a precise temporal program. Consequently, we are analyzing the VMP32 gene expression and regulation in vivo by P-mediated trans-

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Fig. 5. Southern blot analysis of genomic DNA from Canton S (lane 11, aboiubo (lanes 2 and 41, and aboiuboGn (lane 3) females, digested with PstliEcoRl and hybridized with the HindIIIiEcoRl fragment of phage E3 identified by the subclone called pA2 in Figure 4. As was expected from the restriction maps, these digestions give in the abo strains (lane 2 and 4)a 3.5 kb and in the wild-type strain (lane 1)a 6.1 kb fragment. The restriction pattern of the aboluboGn line (lane 31, in homozygous conditions for several generations and having an abo’ phenotype, is identical to that of wild-type.

formation (Spradling and Rubin, 1982; Rubin and Spradling, 1982; Steller and Pirrotta, 1985). The discovery of a VMP gene in region 32E is one example of the new insight provided by the molecular approach. Naturally, our goal is to correlate the genes we isolated molecularly with those identified by genetic analysis. We are unable as yet to associate this gene with any of the mutations we are studying in region 32, which are all semisterile and, in principle, justify their identification as VMP genes.

The Putative abo Gene(s?) Phage E3 was the one on which we identified, by the restriction enzyme site polymorphism approach, a DNA rearrangement present only in stocks carrying the abo mutation (Lavorgna et al., 1989). By Southern blot analysis, using DNAs from homozygous or heterozygous mutant abo, dal, wdl, and hup flies and from Canton S and Oregon R wild-type stocks digested with restriction enzymes and probed with the single isolated

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Fig. 6. Southern blot analysis of genomic DNA from 24 single aboiabo females collected from the abolCy stock, digested with HindIIIiEcoRl and hybridized with the pA2 subclone from Figure 4. The HindIIIiEcoRl digestions give, only in strains carrying a t least

one abo chromosome, the 4.6 kb band seen in the Figure as well as a faint 4.0 kb band, which is not seen in this gel. The 3.2 kb fragment, present only in strains carrying at least a wild-type chromosome, is not present in any of these flies, which are a sample of the flies tested.

phages, we found several restriction site polymorphisms in the region but just one associated exclusively with the abo mutation. The rearrangement was caused by a DNA insert on the abo chromosome in region 32E, which, by restriction mapping and sequence analysis, was identified as the copia-like blood transposon first identified by Bingham and Chapman (1986) in the white blood ( wb') mutation (Ephrussi and Herold, 1945). Figure 4 shows the restriction map of the transposon present in region 32. We observed a strict correlation between the presence of blood transposon and abo phenotypic expression: Whereas the abo stocks (Fig. 5, lanes 2 and 4)contained this element in region 32E, the wild-type (Fig. 5, lane 1) and certain abo homozygous strains that had lost the abo maternal effect (Fig. 5, lane 3) did not. The phenotype of these lines was, therefore, wild type, and the molecular structure of region 32E was identical with that of the wild-type stocks. In the hope of documenting on a molecular level the loss of the abo phenotype, in new homozygous lines, we 1) followed the abo phenotype by genetic approaches (number of progeny per mother, diagnostic cross, etc.) and 2) analyzed the molecular structure. In some of these lines, we observed the persistence of the abo phenotype up t o the sixth to eighth generations and a sudden disappearance of the maternal effect in the seventh to ninth generations. The pattern observed

with Southern analysis showed that the appearance of the abo' phenotype was accompanied by that of a wild-type molecular pattern (Lavorgna et al., 1989). The results we obtained by analyzing region 32 of the different strains at the molecular level seemed to indicate that, in those lines showing loss of the abo phenotype, a reversion to the wild-type DNA structure occurred and, therefore, recovery of the abo mutation from the reverted strains was unlikely. From these homozygous lines, we constructed heterozygous aboiCy lines but so far (G 42) without observing the reappearance of the abo phenotype in homozygous females from these heterozygous strains, in contrast with previous reports (Krider and Levine, 1975). While following, in abo homozygous lines, the abo phenotype at each generation in parallel with the molecular analysis, we found that there are some homozygous lines in which loss of the phenotype is not accompanied by loss of the blood transposon (unpublished results). This fact, together with the observation of different patterns shown by these lines in reaching the wild-type phenotype, seems to confirm that this phenomenon can follow different pathways. The excision of the transposon obviously confers such a selective advantage in homozygous condition that the flies in which the event occurs very rapidly substitute completely the abo flies. The question of whether the transposon is lost in the heterozygous strains was

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Fig. 7. DAPI staining of stage 13egg chambers from homozygous hup females (A) compared with those of wild-type females (B).

MATERNAL-EFFECT MUTANTS IN DROSOPHILA addressed by analyzing single homozygous flies from the heterozygous abo stock. The HindIII/EcoRl digestions of genomic DNA from adult flies, only in strains carrying at least one abo chromosome, due to the presence of the transposon in region 32E, show a 4.6 kb band as well as a faint 4.0 kb band, whereas, in strains carrying at least a wild-type chromosome, they show a 3.2 kb fragment (Lavorgna et al., 1989). Figure 6 reports the HindIII/EcoRl restriction pattern of DNA extracted from a sample of single flies as evidenced by the wild-type diagnostic probe for the presence of the transposon. As can be seen, all the flies carry the 4.6 kb band and, therefore, the transposon in region 32E. Apart from having identified a transposon moving with a high frequency under controlled genetic conditions, this finding can be considered as the starting point for the identification of the abo gene. We are studying the transcripts identified by genomic and cDNA clones near the transposon insertion site and looking for differences in the RNA profile between the wild-type and abo stocks. By Northern analysis, we have identified transcripts with genomic probes on both sides of the transposon and found quantitative and qualitative differences in the transcription patterns between the wild-type and abo stocks (unpublished results).

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ent approaches described lead to the hypothesis that they belong to a group of hypomorphic mutations of genes whose products are necessary in many developmental stages and whose absence would be lethal for the flies. The following findings-1) the phenotypic characteristics showing also a zygotic semilethality and, in some cases, morphological alterations in the adult structures, besides the maternal effect; 2) the pattern obtained with the DAPI staining of the ovaries and early embryos; 3) the sequence homology of some cDNAs correlated with some of the mutations; and 4) the distribution of some transcripts in “in situ” hybridization experiments-appear to indicate that this group of genes is involved in very general processes correlated with cell proliferation and/or cell migration. Possibly, the different genes are implicated in different aspects of these processes and in different tissues and developmental stages.

ACKNOWLEDGMENTS

We thank all the other members of our groups, G. Gargiulo, S. Gigliotti, G. Lavorgna, L. De Ponti, M.R. Santo, and A.M. Rosica, for their invaluable contribution in following the various lines in the research analysis. We also thank Mr. Enzo De Falco for his technical assistance and Mrs. Silvia Andone, our stock curator. We are particularly grateful to Mrs. Susan Cellular Phenotypes A detailed study is in progress on the structure of the Hafkin for her editorial work. The different lines in this ovaries and early embryos produced by homozygous work were supported in part by grants from CNR, Italy hup, wdl, dal, and abo mutant females using the DAPI (Progetti Finalizzati: Biotecnologie e Biostrumentazistaining method (Coleman et al., 1981; Bansel and one, Ingegneria Genetica), and are part of a FAOIIAEA Pferiffer, 1985). Notwithstanding an overall alteration Agreement (No. 5116/CF). in the wdl, dal, and abo ovary structures, the few egg REFERENCES chambers produced by homozygous mutant females seem normal. When observing the egg chambers from Bansel R, Pferiffer SE (1985): Developmental expression of 2‘-3-cyclic nucleotide 3’-phosphohydrolase in dissociated fetal rat brain culhomozygous hup females (Fig. 7B), we found a signifitures and rat brain. J Neurosci Res 14:2134. cant alteration in the distribution and number of T, Pachi C, Gergen JP, Wensink PC (1980):The isolation and follicle cells and in the appendix structure of egg Barnet characterization of Drosophila yolk protein genes. Cell 21:729-736. chambers in the late stages (11-14) compared with Bellen HJ, OKane CJ, Wilson C, Grossniklaus U, Pearson RK, those of wild-type females (Fig. 7A). Since earlier egg Gehring W (1989): P-element-mediated enhancer detection: A versatile method to study development in Drosophila. Genes Dev chamber stages, when follicle cells divide, appear t o be 3:1288-1300. normal, the alteration does not seem to concern steps in Bingham PM, Chapman CH (1986): Evidence that white-bloodis a cell division but rather to involve cell migration, cell novel type of temperature-sensitive mutation resulting from temfusion, or cell death. Results emerging from the obserperature-dependent effects of a transposon insertion on formation of white transcript. EMBO J 5:3343-3351. vation of early hup, abo, and dal embryos we are also trying to analyze in detail. Early hup embryos show the Brennan MD, Weiner AJ, Goralsky TJ, Mahowald AP (1982): The follicle cells are a major site of vitellogenin synthesis in D. melasame strong alteration in the pattern of nuclei distrinogaster. Dev Biol 89:225-236. bution, and 10% of the embryos seem to stop develop- Burke T, Waring GL, Popodi E, Minoo P (1987): Characterization and sequence of follicle cell genes selectively expressed during vitelline ment immediately after the fertilization. Concerning membrane formation in Drosophila. Dev Biol 124:441-450. the dal embryos, it is already known (Sullivan et al., Coleman AW, Maguire MJ, Coleman JR (1981): Mithramycin and 1989) that this phenotype results from the products of 4’-6-diamidino-2-phenylindole DNA staining for fluorescence miabnormal nuclear cortical division sinking inward. crospectro photometric measurement of DNA in nuclei, plastids and

CONCLUSIONS Notwithstanding the importance of the mutants under study at the genetic and molecular levels, their role during oogenesis and early embryogenesis is still unknown. However, the data obtained with all the differ-

virus particles. J Histochem Cytochem 29:959-964. Ephrussi B, Herold J L (1945): Studies of eye pigments of Drosophila. 11. Effect of temperature on the red and brown pigments in the mutant blood. Genetics 30:62-70. Erdelyi M, Szabad J (1989): Isolation and characterization of dominant female sterile mutations of D. melanogaster. I. Mutations on the third chromosome. Genetics 122:lll-127.

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