Oogenesis in the female sterile (1)42 mutant of Drosophila melanogaster.

The sex-linked mutation fs(1)42 was induced by ethyl methane sulfonate. It has no effect on either the external morphology or longevity of adult hemizygotes or homozygotes. Heterozygotes and hemizygotes are fertile, but homozygotes are sterile. Egg chamber development proceeds through stages 8, and thereafter chambers degenerate. Dying follicle cells are seen in chambers at all positions in the ovarioles. Profollicle cells also die within germaria, and clusters of sister cystocytes take longer than normal to receive their coverings of follicle cells. Egg chambers in the vitellarium contain only about 60% the normal number of follicle cells, these generally have greater lateral dimensions, and their nuclei and nucleoli are also larger than normal. The follicular envelope of mutant chambers often contains gaps through which cystocytes send cytoplasmic projections. Abnormalities seen in development of the fs(1)42 oocyte are likely to be due to its envelope of defective follicle cells.     

RotundRacGAP functions with Ras during spermatogenesis and retinal differentiation in Drosophila melanogaster

Our analysis of rotund (rn) null mutations in Drosophila melanogaster revealed that deletion of the rn locus affects both spermatid and retinal differentiation. In the male reproductive system, the absence of RnRacGAP induced small testes, empty seminal vesicles, short testicular cysts, reduced amounts of interspermatid membrane, the absence of individualization complexes, and incomplete mitochondrial condensation. Flagellar growth continued within the short rn null cysts to produce large bulbous terminations of intertwined mature flagella. Organization of the retina was also severely perturbed as evidenced by grossly misshapen ommatidia containing reduced numbers of photoreceptor and pigment cells. These morphological phenotypes were rescued by genomic rnRacGAP transgenes, demonstrating that RnRacGAP function is critical to spermatid and retinal differentiation. The testicular phenotypes were suppressed by heterozygous hypomorphic mutations in the Dras1 and drk genes, indicating cross talk between RacGAP-regulated signaling and that of the Ras pathway. The observed genetic interactions are consistent with a model in which Rac signaling is activated by Ras and negatively regulated by RnRacGAP during spermatid differentiation. RnRacGAP and Ras cross talk also operated during retinal differentiation; however, while the heterozygous hypomorphic drk mutation continued to act as a suppressor of the rn null mutation, the heterozygous hypomorphic Dras1 mutation induced novel retinal phenotypes.     

Characterization of the female sterile (1) 1304 mutant of Drosophila melanogaster: pattern of RNA metabolism in the ovary

The female sterile mutant of Drosophila melanogaster, fs(1)1304 (1-19 +/- 2), has been characterized. Our studies show that the mutation affects the organization of nucleolar material in the ovarian nurse cells and the pattern of RNA metabolism in the ovary. Autoradiographic analysis of incorporation of 3H-uridine in vivo and analysis of 3H-uridine incorporation into high molecular weight RNA in vitro suggest that RNA from the ovaries of homozygous fs flies is degraded at a higher rate than that from heterozygous fs and wild-type ovaries. It is likely that the RNA class affected is ribosomal RNA. These data are discussed in the context of the functional role for the wild-type gene allelic to fs(1)1304, and it is suggested that one of the effects of the mutation may be on the biogenesis of ribosomes that are to be stored in the oocyte.     

Y-Linked male sterile mutations induced by P element in Drosophila melanogaster.

The Y chromosome in Drosophila melanogaster is composed of highly repetitive sequences and is essential only in the male germ line. We employed P-element insertional mutagenesis to induce male sterile mutations in the Y chromosome. By using a combination of two modifiers of position effect variegation, adding an extra Y chromosome and increasing temperature, we isolated 61 P(ry+) elements in the Y chromosome. Six of these Y-linked insertions (approximately 10%) induced male sterile mutations that are mapped to two genes on the long and one on the short arms of the Y chromosome. These mutations are revertible to the wild type in a cell-autonomous and germ-line-dependent manner, consistent with previously defined Y-linked gene functions. Phenotypes associated with these P-induced mutations are similar to those resulting from deletions of the Y chromosome regions corresponding to the male fertility genes. Three alleles of the kl-3 gene on the Y long arm result in loss of the axonemal outer dynein arms in the spermatid tail, while three ks-2 alleles on the Y short arm induce defects at early postmeiotic stages. The recovery of the ms(Y) mutations induced by single P-element insertions will facilitate our effort to understand the structural and functional properties of the Y chromosome.     

Microsporogenesis in male sterile Rosmarinus officinalis L. (Lamiaceae), an ultrastructural study

Within an extensive reproductive biology program on Rosmarinus officinalis a study of microsporogenesis in male sterile plants has been carried out. Two events, related to the two types of male sterile flowers found in this species (MS; Male sterile and INT; Intermediate male sterile flowers), are described. The first event is characterized by the early appearance of necrotic areas in the anther tissues during the final differentiation phases of the MS flowers, before meiosis takes place. Initially, these necrotic areas are small, later on they enlarge affecting the sporogenous tissue and tapetum. All the anthers in which necrosis has begun finally become aborted and lack pollen grains. The second event is manifested in the anthers of the INT flowers. Previous to the release of the microspores from the callosic envelope, vacuolisation of the tapetal cells takes place. The tapetum does not properly carry out its secretory function. It accumulates some substances in vacuoles and starch granules in plastids. The microspores degenerate in the vacuolate stage. In the epidermal and endothecial cells modifications are observed which may be related to the carbohydrate metabolism. Indehiscent, whitish anthers containing inviable microspores result. We have not found significant differences between the mitochondria in the anthers of the fertile flowers and MS or INT flowers. But we suggest, for both events, a metabolic failure, possibly controlled by cytoplasmic genes as the origin of male sterility. Paracrystalline material has been detected in microspore nuclei of developing INT anthers, though it does not seem to be related to the expression of male sterility.     

Mutagen sensitivity of Drosophila melanogaster. VI. Evidence from the excision-defective mei-9AT1 mutant for the timing of DNA-repair activity during spermatogenesis

The sex-linked recessive lethal test has been used to compare mutation induction by ethyl methanesulfonate and methyl methanesulfonate in spermatogenic stages of the DNA repair-deficient mei-9AT1 mutant and a repair-proficient control strain. For both agents, the data demonstrate that induced mutation rates are similar in both strains for the meiotic and post-meiotic broods. Conversely, for spermatogonial broods, the data indicate that the excision-deficient strain exhibits a 4-8 fold increase in induced mutation rate in comparison to the excision-proficient control strain. These experiments suggest that the low mutability of gonial cells normally observed for these agents is due to effective excision-repair processes which function until the commencement of meiosis. From alkylation mutagenesis experiments with repair-deficient E. coli strains, we note that the mei-9 strain exhibits pleiotropic mutant phenotypes very similar to those displayed by the uvr D mutant. By analogy with these studies, we speculate that mei-9, like uvr D, is deficient in a DNA unwinding protein.     

gaMS-1: A gametophytic male sterile mutant in maize

Several pollen-specific genes from different species have been isolated and characterized at the molecular level, but the precise role of most of them is unknown. Mutant analysis represents a direct approach to uncovering gene function, but the paucity of available mutants affecting pollen development and/or function and the poor characterization of the known mutants have so far limited the exploitation of this approach. Here we present the cytological characterization ofgametophytic male sterile-1 (gaMS-1), a maize mutant that we identified in a program of transposon insertion mutagenesis for the production of mutations in gametophytically acting genes involved in microsporogenesis.gaMS-1 is expressed during or immediately after the first microspore division and leads to the production of immature, nonfunctional pollen grains. The mutation appears to affect the events leading to the developmental switch that follows the first microspore mitosis.     

An ultrastructural analysis of the ecdysoneless ((l(3)ecd 1ts) ring gland during the third larval instar of Drosophila melanogaster

Summary In the late third larval instar of Drosophila melanogaster, the prothoracic gland, an endocrine portion of the ring gland, synthesizes ecdysteroids at an accelerated rate. The resultant ecdysteroid titer peak initiates the events associated with metamorphosis. The normal prothoracic gland displays several ultrastructural features at this developmental stage that reflect increased steroidogenic activity, including extensive infoldings of the plasma membrane (membrane invaginations) and an increase in both the concentration of smooth endoplasmic reticulum (SER) (or transitional ER) and elongated mitochondria. By contrast, the prothoracic glands of larvae homozygous for a conditional larval lethal mutation, l(3)ecd ^1ts, not only fail to produce ecdysteroids at normal levels at the restrictive temperature (29° C), but also acquire abnormal morphological features that reflect the disruptive effects of the mutation. These abnormalities include an accumulation of lipid droplets presumed to contain sterol precursors of ecdysteroids, a disappearance of SER and a drastic reduction of membrane invaginations in the peripheral area of the cell. These morphological defects are observed in prothoracic glands dissected from larvae transferred from 18° C to 29° C approximately 24 h before observation and also within 4 h of an in vitro transfer to 29° C following dissection from wandering third instar larvae reared at 18° C. No ultrastructural abnormalities were noted in the corpus allatum portion of mutant ring glands. These observations further indicate the direct involvement of the ecd gene product in ecdysteroid synthesis and suggest a role for the gene in the proper transport of precursors to the site where they can be utilized in ecdysteroid biosynthesis.     

Heat shock during the development of brain structures of Drosophila: the memory development in the l(1)ts403 mutant of Drosophila melanogaster

The structures and functions of many genes are homologous in Drosophila and humans. Therefore, studying pathological processes in Drosophila, in particular neurogenerative processes accompanied by progressive memory loss, helps to understand the ethiology of corresponding human disorders and to develop therapeutic strategies. It is believed that the development of neurogenerative diseases might result from alterations in the functioning of the heat shock/chaperone machinery. In view of this, we used Drosophila mutant l(1)ts403 with defective synthesis of heat shock proteins for studying learning and memory in a test of conditioned courtship suppression following a heat shock given at different developmental stages. High learning indices were registered immediately and 30 min after training both in the intact controls and in flies subjected to different developmental heat shocks. This indicated normal learning and memory acquisition in the mutant. At the same time, memory retention (3 h after training) suffered to different extent depending on the developmental stage. The remote effects of heat shock given during the formation of the mushroom bodies indicated the important role of this brain structure in the memory formation. The observed memory defects may result from alterations both in mRNA transport and in the functions of molecular chaperones in the l(1)ts403 mutant.     

Juvenile hormone involvement in Drosophila melanogaster male reproduction as suggested by the Methoprene-tolerant(27) mutant phenotype

Juvenile hormone (JH) involvement in male reproduction is poorly understood. In Drosophila melanogaster adults, JH deficiency has been shown to result in lowered protein synthesis in male accessory glands. To probe additional roles, we have examined males homozygous for a null allele of Methoprene-tolerant (Met). This gene is involved in the action of JH, possibly at the JH receptor level, and Met²⁷ null mutants reflect a diminution of JH action. Met²⁷ males were found to have reduced protein accumulation in male accessory glands and to court and mate wild-type females much less avidly than do either Met⁺ or Met²⁷; Met⁺ transgenic males. Exposure of Met²⁷ males to methoprene partially rescued the courtship deficiency. However, sperm transfer as reflected by fertility of Met²⁷ fathers was found to be similar to that of Met⁺. Taken together with previous work examining the JH-deficient mutant apterous, these results corroborate JH involvement in protein synthesis in the male accessory glands and suggest a role for JH in promoting male mating behavior in these flies.     

Three-dimensional reconstruction of innermost chorion layer of Drosophila grimshawi and Drosophila melanogaster eggshell mutant fs(1)384.

A low-resolution three-dimensional structure of the crystalline innermost chorionic layer (ICL) of the Hawaiian species Drosophila grimshawi and the Drosophila melanogaster eggshell mutant fs(1)384 has been calculated from electron microscope images of tilted negatively stained specimens. The isolated ICL of Drosophila grimshawi is a three-layer structure, about 36 nm thick, whereas the ICL of Drosophila melanogaster eggshell mutant fs(1)384 is a single layer, about 12 nm thick. Each unit in both crystalline structures includes octamers made up of four heterodimers. Crosslinks between the structural elements, both within and between unit cells form an interconnecting network, apparently important in maintaining the integrity of the layer. A model which may account for the ICL self-assembly formation in vivo and the ICL observed lattice polymorphism is proposed, combining data from the three-dimensional reconstruction work and secondary structure features of the ICL component proteins s36 and s38.     

dSETDB1 and SU(VAR)3-9 sequentially function during germline-stem cell differentiation in Drosophila melanogaster

Germline-stem cells (GSCs) produce gametes and are thus true "immortal stem cells". In Drosophila ovaries, GSCs divide asymmetrically to produce daughter GSCs and cystoblasts, and the latter differentiate into germline cysts. Here we show that the histone-lysine methyltransferase dSETDB1, located in pericentric heterochromatin, catalyzes H3-K9 trimethylation in GSCs and their immediate descendants. As germline cysts differentiate into egg chambers, the dSETDB1 function is gradually taken over by another H3-K9-specific methyltransferase, SU(VAR)3-9. Loss-of-function mutations in dsetdb1 or Su(var)3-9 abolish both H3K9me3 and heterochromatin protein-1 (HP1) signals from the anterior germarium and the developing egg chambers, respectively, and cause localization of H3K9me3 away from DNA-dense regions in most posterior germarium cells. These results indicate that dSETDB1 and SU(VAR)3-9 act together with distinct roles during oogenesis, with dsetdb1 being of particular importance due to its GSC-specific function and more severe mutant phenotype.     

A histological and ultrastructural analysis of developmental defects produced by the mutation,lethal(1)myospheroid,in Drosophila melanogaster

The recessive embryonic lethal, lethal(1)myospheroid, is located at 21.7 map units on the X chromosome in Drosophila melanogaster. Embryos hemizygous for this mutation appear to develop normally until the time of the first muscular contractions. Due to the physical stress of these initial contractions, dramatic tissue separations occur, which characterize the phenotype of this mutation. The dorsal suture separates with the herniation of midgut and nervous tissue, and the somatic and visceral muscles retract from their sites of attachment. An ultrastructural examination of the development of the muscle attachment sites in these embryos indicates that in 1(1)mys embryos there is a delay in the formation of normal cell-cell attachments. At the muscle-tendon cell junction, the deposition of the apparently normal extracellular matrix of this desmosomal attachment occurs considerably later in development than normal. The 1(1)mys locus apparently makes a product which is either defective, made more slowly, or produced in smaller amounts than normal. The 1(1)mys product is probably necessary for the production of a component of the extracellular matrix of cell-cell attachments.