Restoration of fertility in sterilized Drosophila eggs by transplantation of polar cytoplasm

A technique for transplantation of cytoplasm between Drosophila eggs is described. Polar cytoplasm of newly laid eggs was first made ineffective for the determination of germ cells by UV irradiation. The sterility which results from this UV irradiation could be prevented by the injection of polar cytoplasm, but not by the injection of anterior cytoplasm from unirradiated donor eggs. The results provide the first demonstration of transplantation of agents causing determination in an insect and also provide a bioassay for these agents.Microscopical observations of UV irradiated eggs with and without transplanted polar cytoplasm revealed that UV irradiation delays the migration of cleavage nuclei into the posterior periplasm and prevents cytoplasmic protrusions at the posterior pole from becoming isolated from the periplasm to form pole cells. Transplantation of polar cytoplasm repairs these defects.     

Adherens junction remodeling by the Notch pathway in Drosophila melanogaster oogenesis

Identifying genes involved in the control of adherens junction (AJ) remodeling is essential to understanding epithelial morphogenesis. During follicular epithelium development in Drosophila melanogaster, the main body follicular cells (MBFCs) are displaced toward the oocyte and become columnar. Concomitantly, the stretched cells (StCs) become squamous and flatten around the nurse cells. By monitoring the expression of epithelial cadherin and Armadillo, I have discovered that the rate of AJ disassembly between the StCs is affected in follicles with somatic clones mutant for fringe or Delta and Serrate. This results in abnormal StC flattening and delayed MBFC displacement. Additionally, accumulation of the myosin II heavy chain Zipper is delayed at the AJs that require disassembly. Together, my results demonstrate that the Notch pathway controls AJ remodeling between the StCs and that this role is crucial for the timing of MBFC displacement and StC flattening. This provides new evidence that Notch, besides playing a key role in cell differentiation, also controls cell morphogenesis.     

Social modulation of oogenesis and egg laying in Drosophila melanogaster

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A cytological and genetic study of oogenesis in Drosophila melanogaster

A newly derived collection of 98 autosomal recessive female-sterile (fs) lines induced by ethyl methanesulfonate (EMS) in Drosophila melanogaster has been studied genetically and cytologically. By the use of phenotype and complementation tests, the 98 fs lines were resolved into 19 fs genes on the 2nd chromosome and 17 fs genes on the 3rd chromosome. Nearly half of the fs lines turned out to be replicates of noninduced fs mutant loci that preexisted in the EMS-treated files.Systematizing these fs genes according to their phenotypic and morphological defects focuses attention on their relevance to five major aspects of oogenesis: (1) the developmental organization of the ovary, (2) the synthesis and deposition of yolk material, (3) the formation of the chorion, (4) the control of egg-laying, and (5) the construction of the internal milieu of the ovarian oocyte for normal embryogenesis.The results of this study demonstrate that a systematic collection and characterization of fs mutants can provide the genetic tools needed to study the complex interactions which proceed undetected during normal oogenesis.     

Repair of UV-induced pyrimidine dimers in the individual genes Gart, Notch and white from Drosophila melanogaster cell lines.

The excision repair of UV-induced pyrimidine dimers was investigated in three genes: Gart, Notch and white in a permanent Drosophila cell line Kc, derived from wild type Drosophila melanogaster embryonic cells. In this cell line Gart and Notch are actively transcribed, whereas white is not expressed. In all three genes UV-induced pyrimidine dimers were removed with the same rate and to the same extent: 60% removal within 16 hours, up to 80-100% in 24 hours after irradiation with 10 or 15 J/m2 UV. These kinetics are similar to the time course of dimer removal measured in the genome overall. No difference in repair of the inactive white locus compared to the active Gart and Notch genes was found. Similar results were obtained using a different wild type cell line, SL2, although repair appeared to be somewhat slower in this cell line. The results are discussed with respect to the data found for gene specific repair in other eukaryotic systems.     

Associations between female remating behavior, oogenesis and oviposition in Drosophila melanogaster and Drosophila pseudoobscura

An association between female remating behavior, oogenesis and oviposition was examined in Drosophila melanogaster and Drosophila pseudoobscura to investigate mechanisms that elicit remating. Females receptive to remating oviposited more eggs in both species; however, the species differed in the association between remating behavior and the number and distribution of oocyte stages. We found no differences in the number of either developing eggs of different stages or mature eggs between female D. pseudoobscura that were either receptive or nonreceptive to remating. In contrast, D. melanogaster females that are receptive to remating had significantly more mature eggs in the ovaries than nonreceptive females. Nonremating females had a significantly greater number of immature, vitellogenic oocytes. These results suggest that factors associated with oogenesis are related to female remating behavior in D. melanogaster but not in D. pseudoobscura. We discuss these results in conjunction with other evidence on the role male ejaculatory components play in mediating female remating behavior.     

Developmental analysis of lipids from wild-type and adipose60 mutants of Drosophila melanogaster

A comparative developmental analysis was made of lipids from wild-type and adipose60 (adp60) mutants of Drosophila melanogaster. The lipid content and fatty acid profiles of late third instar larvae, pupae, and mature adults were characterized in methanol:chloroform extracts utilizing thin layer and gas-liquid chromatography. Total lipid content of mutant adults was approximately twice that of the wild-type, but no genotypic differences in lipid content were seen in earlier developmental stages. No sexual dimorphism was observed in total lipid content, although fatty acid profiles revealed some sexual differences. Many stage-specific differences in fatty acid profiles and lipid content were developmentally associated with each genotype. Mutants tended to retain the larval phenotype in lipid content and, to a lesser extent, in fatty acid profile. In comparison to wild-type, mutants tended to have increased lipid saturation, especially in 16-carbon fatty acids in mature adults and in 18:0 fatty acids in late larvae and pupae. No significant difference between the mutants and wild-type appeared in the developmental profiles for 14:1 fatty acid isomers. Hence, adp60 does not alter the desaturation-elongation pathway, a secondary pathway for fatty acid desaturation in Drosophila, which received support from this analysis.     

Tyrosine and catecholamine metabolism in wild-type Drosophila melanogaster and a mutant, ebony

Microinjection of radioactive tyrosine, dopa, and dopamine into mature larvae of Drosophila revealed that the sclerotization pathway is similar but not identical to that in Calliphora: (a) tyrosine is converted to tyrosine-o-phosphate and not to dopa, and (b) the substrate N-acetyldopamine does not accumulate.Larvae of the mutant ebony appear to be similar to the wild type with respect to tyrosine, dopa, and dopamine utilization. About the time of eclosion, however, ebony has twice as much dopamine as normal. Some implications of this are discussed with reference to the mutant phenotype.     

Dosage of 5 S and ribosomal genes during oogenesis of Drosophila melanogaster

During growth, the Drosophila egg chamber increases its DNA content over a thousandfold, mainly by polyploidization of the nurse cell nuclei. We wanted to determine if 5 S and ribosomal genes are replicated to the same extent as the remaining DNA. Egg chambers were mass fractionated to represent different size classes and, therefore, different stages of oogenesis. Nucleic acids were extracted from each class of egg chambers, and after removal and quantitation of the RNA, the content of 5 S and ribosomal genes in the different DNA fractions was assayed by filter hybridization. Diploid DNA and DNA from polytene salivary gland cells served as references. It was concluded that: (1) Ribosomal genes become underreplicated as oogenesis proceeds, but to a much lower extent than in polytene chromosomes of salivary glands of the same organism. (2) By contrast, 5 S genes are equally replicated in egg chambers of all stages of oogenesis. (3) Notwithstanding the large increase in DNA content of egg chambers during oogenesis, the increase in total RNA content (mostly ribosomal RNA) is over 15 times as large.     

Two discrete modes of histone gene expression during oogenesis in Drosophila melanogaster

We have used in situ hybridization to ovarian tissue sections to study the pattern of histone gene expression during oogenesis in Drosophila melanogaster. Our studies suggest that there are two distinct phases of histone gene expression during oogenesis. In the first phase, which occurs during early to middle oogenesis (stages 5-10A), we observe a mosaic pattern of histone mRNA in the 15 nurse cells of the egg chamber: some cells have very high levels of mRNA, while others have little or no mRNA. Our analysis suggests that there is a cyclic accumulation and subsequent degradation of histone mRNA in the egg chamber and that very little histone mRNA is transported into the growing oocyte. Moreover, since the endomitotic replication cycles of the nurse cells are asynchronous during this period, the mosaic distribution of histone message would suggest that the expression of the histone genes in each nurse cell nucleus is probably coupled to DNA replication as in most somatic cells. The second phase begins at stage 10B. During this period, histone gene expression appears to be "induced" in all 15 nurse cells of the egg chamber, and instead of a mosaic pattern, high levels of histone mRNA are found in all cells. Unlike the earlier phase, this expression is apparently uncoupled from the endomitotic replication of the nurse cells (which are completed by the end of stage 10A). Moreover, much of the newly synthesized histone mRNA is transported from the nurse cells into the oocyte where it accumulates and is stored for use during early embryogenesis. Finally, we have also observed tightly clustered grains within nurse cell nuclei in non-denatured tissue sections. As was the case with cytoplasmic histone mRNA, there is a mosaic distribution of nuclear grains from stages 5 to 10A, while at stage 10B, virtually all nurse cell nuclei have grain clusters. These grain clusters appear to be due to the hybridization of nurse cell histone gene DNA to our probe, and are localized in specific regions of the nucleus.     

Partial rescue of dorsal, a maternal effect mutation affecting the dorso-ventral pattern of the Drosophila embryo, by the injection of wild-type cytoplasm

Mutant alleles at the maternal effect locus dorsal cause a dorsalization of the Drosophila embryo. In extreme mutants, the embryos develop exclusively structures which derive from the dorsal-most region in normal eggs, in less strong phenotypes in addition to dorsal structures, structures normally derived from a dorso-lateral to lateral egg region are formed. Injection of cytoplasm from wild-type embryos into mutant embryos partially restores the dorso-ventral pattern in that injected embryos develop additional structures never formed in uninjected control embryos or embryos injected with mutant cytoplasm. The phenotype of injected embryos resembles that of weaker alleles at the dorsal locus indicating that the wild-type cytoplasm partially rescues the mutant phenotype. The response of the mutant embryos is restricted to the site of injection and occurs only when cytoplasm is injected into the ventral and not into the dorsal side of mutant embryos. The rescuing activity appears to be equally distributed in cleavage stage wild-type embryos, whereas, in syncytial blastoderm embryos, cytoplasm from the ventral side is about twice as effective as that taken from the dorsal side.     

Microcompartmentation of cAMP in wild-type and memory-mutant dunce strains of Drosophila melanogaster

The "enzyme-probe" method [Solti M, Friedrich P: Eur J Biochem 95:551, 1979] has been applied to characterize the cyclic AMP pool in wild-type Canton-S and memory-mutant dunceM11 strains of Drosophila melanogaster. The kinetics of cyclic AMP breakdown in whole fly homogenates by endogenous cyclic nucleotide phosphodiesterase(s) indicate that the cyclic AMP pool is divided into free and bound fractions. The bound fraction in Canton-S and dunceM11 is 0.5 and 1.5 pmole/mg fly, respectively. Considering the total cyclic AMP content of the two strains, 1.6 and 10 pmole/mg fly, respectively, we conclude that the bulk of excess cyclic AMP in the mutant is free nucleotide.     

Proteins shifting from the cytoplasm into the nuclei during early embryogenesis of Drosophila melanogaster

Monoclonal antibodies have been used to study the distribution of several proteins in cleavage and blastoderm stages of Drosophila melanogaster. These antigens are known to be associated with hnRNA-containing particles in tissue culture cells. Protein blotting shows that they are present in the embryo 1 hr after egg deposition. A redistribution from the cytoplasm into the somatic nuclei can be observed during developmental stage $\text{12}\text{13}$, one stage prior to the formation of the cellular blastoderm. Yolk nuclei become stained by these antibodies at about the same time. The shift into pole cell nuclei, however, occurs $\text{1}\text{1}\text{2}$ hr later, during the migration of these cells into the posterior midgut rudiment.     

Translating available food into the number of eggs laid by Drosophila melanogaster

In Drosophila and other insects egg production is related to the nutrients available. Somehow the nutritional status of the environment is translated into hormonal signs that can be "read" by each individual egg chamber, influencing the decision to either develop into an egg or die. We have shown that BR-C is a control gene during oogenesis and that the differential expression of BR-C isoforms plays a key role in controlling whether the fate of the egg chamber is to develop or undergo apoptosis.