Cytophotometric studies on cells from the ovaries of otu mutants of Drosophila melanogaster

Summary Amounts of chromosomal DNA were estimated for Feulgen-stained, ovarian cells from flies carrying certain mutant alleles of the otu (ovarian tumor) gene. Epithelial sheath cells and lumen cells were found to contain the diploid (2C) amount of DNA and therefore served as internal, cytophotometric standards. Mitotically active follicle cells over young tumors-from homozygous otu ¹ females contained either the 2C or 4C amounts of DNA; whereas, the tumor cell population contained 2C, 4C and 8C nuclei and many intermediate values. Egg chambers also occur in homozygous otu ⁷ females. Follicle cells above these oocytes undergo a maximum of four cycles of endomitotic DNA replication. The accompanying nurse cells (PNC) contain polytene chromosomes. These undergo a maximum of 12 endonuclear replication cycles. The PNCs show the expected levels of DNA for the first 6 cycles and the fraction failing to replicate during subsequent cycles may be as small as 10%. Lower than expected levels of DNA were detected in PNCs from an otu ¹/otu ³ ovary, reflecting roughly 20% underreplication. The latter PNCs may have been interrupted before DNA synthesis was concluded. No simple model of genomic underreplication accounts for the several different patterns of DNA behavior observed for various otu mutants.     

Polytene chromosomes from ovarian pseudonurse cells of the Drosophila melanogaster otu mutant

Certain mutant alleles of the otu locus in Drosophila melanogaster produce abnormal nurse cells in the ovaries. These cells are called pseudonurse cells (PNC), since they generate polytene chromosomes instead of endopolyploid ones and do not normally have an oocyte to nurse. The banding pattern of polytene chromosome 3 from the salivary glands (SG) and from PNCs of homozygous otu ¹ females was compared and a detailed photomap of PNC chromosomes with different degrees of polyteny is presented. The banding pattern was found to be strikingly similiar in the two tissues. The puffing pattern of the PNC chromosomes was also studied and the function of the PNC chromosomes is discussed. No constrictions or breaks were found in the PNC chromosomes which seems to indicate that these sites, which are known to be underreplicated in the SG chromosomes, are equally replicated along with the rest of the chromosomes in the PNC nuclei.     

Cytophotometric evidence for the transformation of oocytes into nurse cells in Drosophila melanogaster

A small proportion of ovarian chambers from females homozygous for the otu7 (for ovarian tumor) mutation contain an "oocyte" that in its nuclear morphology resembles a nurse cell. Such transformed oocytes also appear in colchicine-poisoned wild type ovaries. Cytophotometric estimates demonstrate that these oocytes have undergone 3-4 additional DNA replications, but that they lag behind the adjacent nurse cells by an average of 1.3 replication cycles. It follows that, under certain circumstances, the definitive oocyte can switch to the nurse cell developmental pathway and therefore that a mechanism normally exists for preventing the further replication of its DNA. In the case of otu7, oocytes sometimes restart their endocyclic DNA replications and produce paired, polytene, homologous chromosomes.     

Extent of polyteny in the pericentric heterochromatin of polytene chromosomes of pseudonurse cells of otu (ovarian tumor) mutants of Drosophila melanogaster

In the polytene nuclei of germ-line cells (ovarian pseudonurse cells) of Drosophila melanogaster females mutant for otu ¹¹ (ovarian tumor), the pericentric heterochromatin is much more abundant than in somatic salivary gland cells. This is due to the degree of heterochromatin compaction (and consequently the level of underreplication) being lower in the nurse cells than in the salivary gland cells. The lower level of compaction probably results in a very low degree of position effect gene inactivation in the ovarian nurse cells.     

Effect of otu mutations on male fertility and spermatogenesis in Drosophila melanogaster

The 17-ethyl-methyl-sulphonate (EMS) induced female sterile alleles of the ovarian tumour (otu) locus show a wide spectrum of phenotypes and affect various processes of Drosophila oogenesis. These phenotypes have been previously studied in detail, but the exact molecular function of the otu locus in the different processes of oogenesis is only poorly known. To date, no effect of otu mutations have been reported in the males. However, separate species of otu mRNAs are expressed in the testes and the thorax of the adult male, but their role is not known. In this study we analysed the effects of EMS-induced otu mutations on male fertility. We observed that the proportion of totally sterile males is significantly higher in most of the tested otu strains as compared to the wild type. There was a strong correlation between male sterility and severity of impairment in the female phenotype. Spermatogenesis of these semi-sterile strains was analysed by phase contrast microscopy, Hoechst 33258 and Feulgen stain, and by in situ hybridisation with testis-specific probes. No changes which could account for the induction of sterility were recorded and normal amounts of motile sperm were observed in all strains. Sterility turned out to be a consequence of a failure in mating behaviour. The wild type females refused to react to the courtship attempts of the mutant males. We propose two alternative explanations for this. Either the otu locus may play some important role in male somatic tissue, or some germ line function is necessary for correct mating behaviour.     

Nurse cell polytene chromosomes of Drosophila melanogaster otu mutants: Morphological changes accompanying interallelic complementation and position effect variegation

Combinations of certain mutant alleles of the ovarian tumor gene permit the production of viable eggs. Two alleles that behave in this way are otu⁷ and otu¹. Females homozygous for either allele are sterile, and their ovarian nurse cells (NC) contain giant polytene chromosomes of various morphologies. Fertile flies (otu⁺ / otu⁺, otu⁻ / otu⁷, otu⁺ / otu¹¹) have endopolyploid nurse cells with typical dispersed chromosomes. Fertile hybrids (otu⁷ / otu¹¹) produce large numbers of polytene chromosomes comparable to, and often larger than, classic salivary gland (SG) chromosomes. Therefore, these otu hybrids provide a unique system for studying, at the chromosomal level, the activation and expression of genes functioning during oogenesis. The otu gene encodes a long and a short isoform. The normal long isoform appears to be responsible for the dispersion of chromosomes during the endomitotic DNA replications occurring in ovarian NCs. The genetic inactivation of euchromatic genes placed next to pericentric heterochromatin by a chromosomal rearrangement is accompanied by the compaction of corresponding chromosome regions. A comparative study of the manifestation of position-effect variegation for the polytene chromosomes of SG cells and NCs was made using the Dp(1;1)pn2b and Dp(1;f)1337 rearrangements. The percentage frequencies of block formation in the SG and NC nuclei for Dp (1;1) pn2b rearrangement were 92.6% vs. 15.8%, respectively; for Dp(1;f) 1337, these values were 56.8% vs. 9.7%. Therefore heterochromatin belonging to germ line chromosomes is in a configuration that is far less likely to inactivate inserted segments of euchromatin than is heterachromatin from somatic chromosomes. Dev. Genet. 20:163–174. 1997. © 1997 Wiley-Liss, Inc.     

Fertile heteroallelic combinations of mutant alleles of the otu locus of Drosophila melanogaster

Summary This paper describes the ovarian pathologies observed when 108 different heteroallelic combinations were made involving 17 independent mutations at the ovarian tumor (otu) locus. Most of the mutant phenotypes can be explained as graded responses by individual germ cells to different levels of functionally active otu gene product (OGP) synthesized by the mutant cells themselves. The lowest and highest levels of OGP appear to be produced by otu ¹⁰ and otu ¹⁴, respectively. In most heteroallelic ovaries the alleles have additive effects, and hybrid germ cells reach a developmental stage more advanced than the weaker homozygote but less advanced than the stronger homozygote. However, examples of both positive and negative complementation also have been found, and these suggest that the products encoded by different mutant alleles can combine to form dimers or multimers which may be superior or inferior to the homodimers. In flies homozygous for otu ¹¹ most ovarioles contain tumors, but some germ cells are able to develop further than those in otu ¹⁴ homozygotes. This suggests that, while otu ¹¹ produces intermediate levels of OGP, it also produces a second product (which otu ¹⁴ cannot make) that is utilized at the period in oogenesis when development in cells homozygous for otu ¹⁴ is blocked. When otu ¹¹ is combined with any one of eight specific alleles, it allows oocyte/nurse cell syncytia to differentiate that can complete development and undergo embryogenesis, if fertilized. The endopolyploid nurse cells of these hybrids have giant polytene chromosomes, and the presence of GPCs in functionally active, germ-line derived cells provides an interesting new system for experimental study. Analysis of the characteristic ovarian pathologies produced by flies of different genotypes leads to the conclusion that the products of the otu ⁺ gene are utilized during at least six different periods in Drosophila oogenesis.     

Ovarian pathologies generated by various alleles of the otu locus in Drosophila melanogaster

A comparative cytological study was made of oogenesis in flies carrying various mutant alleles of the female sterile gene otu. It resides at 22.7 on the genetic map and within subdivision 7F of the cytological map of the X-chromosome. Each of the five ethyl methane sulfonate-induced mutations observed falls into one of three classes. In class 1, most mutant ovarioles lack germ cells; in class 2, most mutant ovarioles contain tumorous chambers; and in class 3 mutants, chambers occur that possess defective oocytes. The otu² allele belongs to class 1; otu¹ to class 2; and otu³, otu⁴, and otu⁵ to class 3. The mutations have no effects upon female viability or upon the viability and fertility of hemizygous males. Heterozygous females are fertile and have cytologically normal ovaries. In otu⁵ homozygotes, all ovarioles contain egg chambers, but oogenesis is prematurely terminated to produce a pseudo-stage 12 oocyte. Ovarioles from otu³ and from otu⁴ homozygotes contain both ovarian tumors and oocytes. Pseudonurse cells (PNC), which are cystocytes that have stopped dividing and have entered the nurse cell mode of development, are also abundant. PNCs contain polytene chromosomes. Since the homologs are paired, each nucleus has the haploid number of chromosomes. In chambers lacking an oocyte, the number of PNCs is less than the normal number of nurse cells. In chambers containing an oocyte, the number of accompanying nurse cells may be 15, or above or below normal. In vitellogenic chambers, the chromosomes in the nurse cells connected directly to the oocyte are more expanded than those in more distant nurse cells. The KA14 deficiency lacks the plus allele of otu. KA14 heterozygotes are fertile and have cytologically normal ovaries. When females carry KA14 and otu¹, otu³, otu⁴, or otu⁵, 80% of their ovarioles are agametic. When females carry otu² and one of the other mutant alleles, the ovarioles proceed further in development. So otu² produces a product that has a beneficial effect on the test allele. When two different otu alleles are combined in a single fly, the phenotype of the hybrid ovary usually most resembles that of the ovary homozygous for the “stronger” allele (the otu mutant that allows oogenesis to proceed farthest). The results indicate that the product of the otu⁺ locus functions at least three different times during oogenesis; first to permit oogonia to proliferate, second to control the division and differentiation of germarial cystocytes, and third to facilitate the normal growth of the ooplasm. The gene product appears to be required in higher concentrations at each developmental period. The lesions produced by the mutations are thought to interfere with the stability or functioning of the gene product, and the ovarian phenotype produced by a given genotype depends upon the concentration of functional gene product available to the germ cells.     

Specific DNA sequences are associated with nuclear envelopes of pseudonurse cells in Drosophila melanogaster otu 11

Nuclei of ovarian pseudonurse cells from the mutant strain of Drosophila melanogaster otu 11 are suitable for mapping the attachment of chromosomes to the nuclear envelope (NE). Loci in contact with the NE included region 20CD of the X chromosome, region 41 of chromosome 2, the proximal end of region 81 of chromosome 3, and region 101 of chromosome 4. In situ hybridization revealed that all 4 regions contained sequences homologous to clone lambda20p1.4. DNA of clone lambda20p1.4 was previously found to bind specifically to purified D. melanogaster lamins. These results suggest that specific DNA sequences are involved in attachment of chromosomes to NE in vivo.     

Structural and biochemical studies on four sex-linked chorion mutants of Drosophila melanogaster

Four female-sterile mutants, fs(1)K451, fs(1)K1214, fs(1)K575TS, and fs(1)384, were studied in terms of chorion structure and chorion protein composition. The first three of these mutants cause morphological defects, ie, substantial underproduction and disruption of the endochorion, correlated with underproduction of the six major chorion proteins, s15-s38; the phenotypes are consistent with the observation that these mutants interfere with amplification of the major chorion genes that encode the s15-s38 proteins [Orr et al.: Proc Natl Acad Sci USA 81:3773-3777, 1984; Komitopoulou et al.: Dev Genet 7:75-80, 1986]. The fourth mutant, fs(1)384, and its alleles do not interfere with production of the major chorion proteins and the morphologically detectable bulk of the endochorion but lead to failure of endochorionic organization. Apparently this complementation group is responsible for a minor chorion product, which is evidently important morphogenetically and which is processed posttranslationally in a complex manner [Bauer and Waring: Dev Biol 121:349-358, 1987].     

Cytophotometric DNA determinations and autoradiographic studies in salivary gland nuclei from larvae with different karyotypes in Drosophila melanogaster

Cytophotometric DNA determinations in Feulgen stained mitotic diploid chromosome sets of neuroblasts from larvae of Drosophila melanogaster stocks, which possess different karyotypes, show significant differences between the 4C values, caused by an additional or deficient X- and Y-chromosome depending on the karyotype. The ranges of polytenic DNA size classes are theoretically expected to be doublings of the corresponding 4C mean value of each karyotype. The extinction integral data of nuclei with completely duplicated 4C quantities exclusively fall into the range of the expected size classes. Not all data falling into the range of a size class necessarily originate from duplicated nuclei, because the limits of the DNA size classes cannot be determined by measurements, but must be estimated from the confidence limits of the corresponding 4C mean value. The validity of the mitotic 4C values of the karyotypes X/X and X/Y is tested using data from non-labeled interphase nuclei, where extinction integral data accumulate in two groups. The larger values (= G₂-nuclei) confirm the 4C values of mitotic chromosome sets, and the lower values (= G₁-nuclei) are just half of these. Extinction integrals from individual, ³H-thymidine non-incorporating polytene salivary gland nuclei accumulate in distinct, non-overlapping groups which are always complete doublings of the preceding smaller group. In each karyotype, the most frequent data of each group are in accord with the 4C doublings. The data from labeled nuclei alternate with those from unlabeled nuclei. The measured DNA values of individual polytene nuclei that did not incorporate any ³H-thymidine, demonstrate that all chromosomal DNA replicates completely during polytenization of the chromosomes in the larval salivary gland nuclei of Drosophila melanogaster. Specifically, this would mean that the heterochromatic Y-chromosome replicates as well as the partially heterochromatic X-chromosome along with the autosomes. There is no indication of underreplicating heterochromatin.