Germ line imprinting in Drosophila: Epigenetics in search of function

Germ line imprinting produces parent-specific differences in the behavior of chromosomes or expression of genes. Epigenetic marks, placed on chromosomes in the parental germ line, govern classical imprinted effects such as chromosomal inactivation, chromosome elimination and mono-allelic expression. Germ line imprinting occurs in insects, plants and mammals. Several Drosophila systems display imprinted effects. In spite of this, many aspects of imprinting in flies, including the normal function of this process, remain mysterious. Transgenerational inheritance of epigenetic marks is a powerful force in genome regulation. Elucidation of the mechanism of imprint establishment and maintenance in a model organism, such as Drosophila, is thus of great interest. In this review we summarize the primary systems that have been used to study imprinting in flies and speculate on the origin and biological function of imprinting in Drosophila.     

Germ granules in Drosophila

Germ granules are hallmarks of all germ cells. Early ultrastructural studies in Drosophila first described these membraneless granules in the oocyte and early embryo as filled with amorphous to fibrillar material mixed with RNA. Genetic studies identified key protein components and specific mRNAs that regulate germ cell‐specific functions. More recently these ultrastructural studies have been complemented by biophysical analysis describing germ granules as phase‐transitioned condensates. In this review, we provide an overview that connects the composition of germ granules with their function in controlling germ cell specification, formation and migration, and illuminate these mysterious condensates as the gatekeepers of the next generation.     

Dosage dependence of maternal contribution to somatic cell division in Drosophila melanogaster.

Most mitotic mutants in Drosophila do not lead to lethality in early development despite the highly abnormal chromosome behaviour that they elicit. This has been explained as being the effect of maternally provided wild-type products. We have tested this hypothesis by studying cuticular clones derived from cells in which there has been loss of a marked Y chromosome due to chromosome nondisjunction in individuals homozygous for the mutation abnormal spindle who are progeny of heterozygous mothers. We have found that the size and frequency of these clones are higher than in control flies. Furthermore, by analysing flies whose female parents have different doses of the asp+ gene, we have found that there is a correlation between the amount of maternally contributed asp+ product and the frequency and size of cuticular clones. We have also estimated the time in development when the first mitotic mistakes take place, i.e. the time when maternal products are no longer sufficient to carry out normal cell division.     

A role for the deep orange and carnation eye color genes in lysosomal delivery in Drosophila.

Deep orange and carnation are two of the classic eye color genes in Drosophila. Here, we demonstrate that Deep orange is part of a protein complex that localizes to endosomal compartments. A second component of this complex is Carnation, a homolog of Sec1p-like regulators of membrane fusion. Because complete loss of deep orange function is lethal, the role of this complex in intracellular trafficking was analyzed in deep orange mutant clones. Retinal cells devoid of deep orange function completely lacked pigmentation and exhibited exaggerated multivesicular structures. Furthermore, a defect in endocytic trafficking was visualized in developing photoreceptor cells. These results provide direct evidence that eye color mutations of the granule group also disrupt vesicular trafficking to lysosomes.     

Germ line stem cell differentiation in Drosophila requires gap junctions and proceeds via an intermediate state

Gap junctions coordinate processes ranging from muscle contraction to ovarian follicle development. Here we show that the gap junction protein Zero population growth (Zpg) is required for germ cell differentiation in the Drosophila ovary. In the absence of Zpg the stem cell daughter destined to differentiate dies. The zpg phenotype is novel, and we used this phenotype to genetically dissect the process of stem cell maintenance and differentiation. Our findings suggest that germ line stem cells differentiate upon losing contact with their niche, that gap junction mediated cell-cell interactions are required for germ cell differentiation, and that in Drosophila germ line stem cell differentiation to a cystoblast is gradual.     

Localized defects of blastoderm formation in maternal effect mutants of Drosophila

In the three maternal effect lethal mutant strains of D. melanogaster described in this report, the homozygous mutant females produce defective eggs that cannot support normal embryonic development. The embryos from these eggs begin to develop for the first 2 hr after fertilization in an apparently normal way, forming a blastula containing a cluster of pole cells at the posterior end and a layer of syncytial blastoderm nuclei. During the subsequent transition from a syncytial to a cellular blastoderm, cell formation in the blastoderm is either partially or totally blocked. In mutant mat(3)1 no blastoderm cells are formed, indicating that there are separate genetic controls for pole cells and blastoderm cells. The other two mutants form an incomplete cellular blastoderm in which certain regions of the blastoderm remain noncellular. The noncellular region in mutant mat(3)3 is on the posterior-dorsal surface, covering about 30% of the total blastoderm. In mutant mat(3)6 blastoderm cells are formed only at the anterior and posterior ends, separated by a noncellular region that covers about 70% of the total blastoderm. The selective effects on blastoderm cell formation in the three mutants emphasize the importance of components present in the egg before fertilization for the transition from a syncytial to a cellular blastoderm.The genes defective in the three mutants are essential only for oogenesis and not for any other period of development, as indicated by a strict dependence of the lethal phenotypes on the maternal genotypes. Heterozygous embryos from the eggs of homozygous mutant females die, whereas homozygous mutant embryos from the eggs of heterozygous females develop into viable adults.One of the mutants, mat(3)3, has a temperature-sensitive phenotype. Homozygous mat(3)3 females maintained at a restrictive temperature of 29°C show the lethal maternal effect. However, at a permissive temperature of 20°C the females produce viable adult progeny. The temperature-sensitive period in mat(3)3 females occurs during the last 12 hr of oogenesis, consistent with the maternal effect phenotype of the mutant.     

Germ line polysomy in the grasshopper Atractomorpha similis

Nineteen Eastern Australian populations of the grasshopper Atractomorpha similis (Acridoidea, Pyrgomorphidae) were sampled and male meiotic chromosomes, as well as some male and female somatic mitoses, were examined. In fourteen of these populations, a proportion of the males were found to carry between one and ten extra copies of a particular autosome, the megameric chromosome (A9). Numbers of extra chromosomes varied between but not within the individual follicles of the testis. The extra chromosomes were not found in somatic tissue. In all, 20% of males from the field were germ-line polysomic and within these males, 91% of germ cells were polysomic. In meiosis, extra copies of A9 present as univalents lagged at anaphase I or II and subsequently formed micronuclei which degenerated early in spermiogenesis. As one extra univalent is the most common polysomic condition in natural populations, this elimination of univalents suggests that most polysomic males produce a large proportion of normal haploid sperm. In laboratory cultures, selection for increased frequency of germ-line polysomy, conducted over four generations, raised the proportion of polysomic males from 23% to 71%. Selection against polysomy reduced its frequency to 5%. These breeding experiments also showed that germ-line polysomy is equally transmissible through both the male and the female parent. Transmission data also suggested that these extra chromosomes can arise de novo, presumably by unequal disjunction in previously diploid lines. A computer model was devised, simulating the effects of repeated non-disjunction over a series of mitotic divisions. The behaviour of this model suggested that the distributions of extra chromosome numbers observed in the laboratory generations most probably resulted from such a series of non-disjunctions, occurring in an initially diploid cell population. It seems, therefore, that the transmission of polysomy occurs through the agency of heritable factors which determine the probability of non-disjunction and thus the accumulation of a particular autosome during a specific series of mitotic divisions in the embryonic germ-line.     

Germ line specific expression of a protein phosphatase Y interacting protein (PPYR1) in Drosophila

PPYR1, the product of the CG15031 gene, was identified as a protein phosphatase Y (PPY) interacting protein in Drosophila melanogaster using a yeast two-hybrid screen. PPYR1 displays a biphasic expression pattern: the maternal protein is abundant in the developing egg chambers and in the early embryos, while the zygotic protein appears later in development and is localized specifically in the testes of the males. The maternal and zygotic gene products differ from each other in their size having apparent molecular masses of 47 and 66 kDa, respectively. The maternal PPYR1 is localized in the cytoplasm of the follicular and nurse cells and is deposited as a ribonucleoprotein complex in the oocyte. In the early embryos, the PPYR1 is distributed evenly, and it gradually diminishes during embryonic development. Zygotic PPYR1 is expressed exclusively in the testes, predominantly in the cytoplasm of the spermatocytes. PPY is localized in the nuclei of the same cells. Our results suggest that PPYR1 has two distinct developmental isoforms: a maternal protein the expression of which is independent of PPY and a zygotic protein which is co-expressed with PPY.     

Homoeosis in Drosophila: Evidence for a maternal effect of the polycomb locus

When heterozygous, dominant mutant alleles of the Polycomb locus are associated with a variety of adult homoeotic effects. Zygotes homozygous for these alleles die as late embryos showing homoeotic transformation of head, thoracic, and abdominal segments. This study shows that embryos homozygous for Pc³ are more extreme than those homozygous for Pc¹ or Pc². Moreover, Pc¹/Pc³ heterozygotes are more extensively transformed if their mothers were Pc³/ + than if they were Pc¹/ +; this effect does not depend on zygotic genetic background and must be maternal in nature. Embryos homozygous for Pc³ are less extreme if they arise from Pc³/ + / + than from Pc³/ + mothers. These results strongly suggest that the Polycomb locus acts maternally as well as zygotically to affect early determinative decisions.     

Germ-line dependence of the maroon-like maternal effect in Drosophila

We have used pole cell transplantations to construct germ-line mosaics for maroon-like (mal), a maternal effect mutation in Drosophila. Such mosaics allow one to determine the cell type in which a gene is active. We find that the maroon-like maternal effect is (1) autonomous to the germ line and (2) dose sensitive in germ-line mosaics. Aldehyde oxidase activity is used as a histological probe to investigate the tissue and temporal distribution of mal⁺ activity in the developing ovary. The adult ovary shows mal⁺ activity in the germ line at all discernible stages of oogenesis but no activity is observed in the mesodermally derived follicle cells. Differential mal⁺ activity is observed even in the ovary of the third-instar larvae.     

Drosophila endosomal proteins hook and deep orange regulate synapse size but not synaptic vesicle recycling

To study the function of endosomes at synapses we analyzed the localization and function of two Drosophila endosomal proteins, Hook and Deep orange (Dor), at the larval neuromuscular junction. Hook, a negative regulator of endocytic trafficking, and Dor, a positive regulator of endocytic trafficking, are highly enriched at synapses, especially close to postsynaptic membranes. Mutations in hook (hk) and dor do not affect synaptic vesicle recycling, as assessed by electrophysiological analysis of synaptic transmission and behavioral studies of double mutants with shi(ts) mutations that alter vesicle recycling. However, hk and dor mutations alter the number of presynaptic varicosities (synapse size) in opposing ways. Synapse size is increased in hk(11) mutants and is decreased in dor(4) mutants. Double mutants for dor and hk show a dor-like phenotype. These effects on synapse size parallel known functions of Hook and Dor in endocytosis and strongly indicate a role for endocytic trafficking in the regulation of synapse size in vivo. Our observations suggest a model in which Hook and Dor function in later stages of endocytosis is essential for regulating synaptic plasma membrane composition but not synaptic vesicle recycling.