Analysis of the Drosophila maternal effect mutant MAT(3)1 by pole cell transplantation experiments

Summary Pole cell transplantations were used to construct germ line mosaics of the Drosophila melanogaster maternal effect mutant mat(3)1. The mutant is of particular interest since the development of embryos derived from homozygous mat(3)1 females is arrested at the pole cell stage. Such embryos form exclusively pole cells and no blastoderm cells. By means of germ line mosaics we could demonstrate the primary target tissue of mutant gene expression. For normal development the mat(3)1 ⁺gene has to be expressed in the germ line. Pole cells formed in defective embryos derived from homozygous mutant mothers were transplanted into normal recipient embryos to test their developmental potential. Heterozygous mat(3)1 pole cells were found to form fertile gametes in both sexes whereas homozygous mat(3)1 pole cells form fertile gametes only in males. The lack of progeny derived from homozygous mat(3)1 donor pole cells in recipient females further demonstrates the germ line autonomy of the mat(3)1 mutation. Pole cells from defective embryos that are transplanted into normal hosts colonize the gonads with the same frequency as donor pole cells derived from normal embryos. This indicates that mat(3)1 derived pole cells are normal with respect to their function as germ cells and that the mat(3)1 mutant might therefore offer a convenient source for the mass isolation of functional pole cells.     

A clonal analysis of the roles of somatic cells and germ line during oogenesis in Drosophila

In order to test whether particular female sterile mutations block functions which normally occur in somatic or germ line derivatives, clones homozygous for each mutation were X-ray induced in heterozygous females. Using the germ line-dependent egg marker, fs(1)K10, it was possible to identify the eggs derived from clones which had been induced in the germ line. Mutations were classified as germ line dependent when these eggs also showed the phenotype associated with the female sterile mutation. Two mutations which caused early abnormalities in oogenesis (fs(1)116, fs(1)1304) were shown to affect germ cells, whereas two mutations which caused egg retention (fs(1)462, fs(1)1001) were somatically dependent. A mutation altering egg dimensions without affecting egg volume (short egg) was also shown to depend on somatic cells in the ovary. With one exception (fs(1)K4), mutations which caused production of fragile, collapsed eggs (fs(1)180, fs(1)473, fs(1)384, and fs(1)1163) were somatically dependent. Patches of mutant fs(1)384 morphology were found in the chorions of the eggs not derived from germ line clones. These patches are interpreted as being caused by homozygous clones in the somatically derived follicle cell epithelium and suggest that fs(1)384 affects processes occurring in these cells during the synthesis of the egg coverings.     

Analysis of the Drosophila female sterile mutation fs(1) 1304 by pole cell transplantation experiments

The Drosophila melanogaster mutant fs(1)1304 is an ovary autonomous female sterile mutant that causes abnormal morphology of the egg. Vitellogenesis proceeds at an abnormally slow rate in homozygous females. We have used pole cell transplantation to construct germ line mosaics in order to determine whether the 1304 defect depends upon the genotype of the germ line cells (oocyte or nurse cells) or the somatic line (follicle cells). We have found that the germ line is the primary target tissue where the mutant gene is expressed.     

Specificity of embryonic lethal mutations in Drosophila analyzed in germ line clones

Summary Only a small fraction of the known mutations causing death to homozygous Drosophila produce gross morphological defects during embryogenesis. We have examined fourteen such loci on the X-chromosome to determine: 1) whether the requirement for their respective activities is restricted to embryogenesis; and 2) whether the embryonic phenotype in mutant embryos is affected by the dosage of wild-type alleles in the mother. For two alleles per locus germ line clones were produced during larval development by irradiating females heterozygous for the lethal mutation and a dominant female sterile (ovo^D). Only one of the 14 loci (armadillo) is required during development of the germ cell to make morphologically normal eggs. Mutations at two other loci, (bazooka and Notch), allow normal oogenesis but cause major reductions in the viability of genetically normal (i.e., heterozygous) progeny. The majority of the loci (11/14) are not required in the germ line for either oogenesis or embryogenesis. However, in three cases (extradenticle, faintoid and lethal myospheroid), germ line homozygosity results in a readily detectible enhancement of embryonic phenotype over that observed in embryos derived from heterozygous mothers still possessing one wild type allele. The same six loci which show the most substantial effects on germ line homozygosity (arm, baz, N, exd, ftd and mys) also show an amelioration of the mutant phenotypes when maternal dosage is increased to wild type levels by using attached-X females. Four of these same loci (arm, baz, N and exd were cell lethal in imaginal discs.     

Follicle cells and germ line cells both affect polarity in dicephalic chimeric follicles of Drosophila

Summary In aberrant egg follicles of the pattern mutant dicephalic (dic) the oocyte is wedged in between two groups of nurse cells, and this condition may give rise to embryos which express anterior traits at both ends. We have analysed the role of the dic genotype of the germ line cells and the surrounding somatic follicle cells in the formation of the dic follicular phenotype. By means of pole cell transplantations into Fs (1) K 1237 hosts (this cell-autonomous mutation causes degeneration of the host's germ line cells early in oogenesis), we constructed chimeras in which either the follicle cells, the germ line cells, or both were homozygous for the dic mutation. In all three combinations the dic phenotype was expressed but not in controls with dic ⁺ in both germ line cells and follicular epithelium. Since follicles with the dic phenotype may be produced if either the germ line cells or the follicle cells lack dic ⁺ gene activity we suggest that cellular interactions between both cell types are required for the correct positioning of the oocyte at the follicle's posterior pole.     

Requirement offlex (femalelethal onX) in the development of the female germ line ofDrosophila melanogaster

Drosophila melanogaster females homozygous forflex, an X-linked recessive mutation, do not survive. Hemizygous males are unaffected. Homozygous embryos appear to lack SXL, the product of theSex-lethal (Sxl) gene, apparently as a result of disruption ofSxl splicing. It is known that bothSxl and its somatic splicing regulators [snf andfl(2)d] also function in the development of the female germ line. For this reason, we investigated the role offlex in the germ line by generatingflex/flex clones inflex/+ females. Females carrying such clones in their germ lines do not lay eggs whereas females carryingflex+ eggs lay viable eggs. Additionally, DAPI staining of ovarioles showed that diploid germ cells that are homozygous mutant forflex do not complete oogenesis. These results indicate that theflex+ gene product may be required for the development of the female germ line.     

Genetic and developmental studies of a new Grandchildless mutant of Drosophila melanogaster

Summary A new grandchildless, maternal-effect temperature-sensitive mutant of Drosophila me anogaster, gs (2) M, was isolated in our laboratory. At 28.5° C, homozygous gs (2) M/gs (2) M females lay a normal number of eggs, but about 20% of them fail to hatch and about 40% die just after hatching. The remaining embryos, which pass through this critical stage, complete their development normally, but some of them are devoid of pole cells and thus produce agametic adults. The death of embryos is maternally determined and the hatching probability of an embryo does not depend on its own genotype. The influence of several factors on the phenotypic expression of the new mutant, e.g., age of the females, temperature and number of generations under homozygous condition, is described. Mutants of the type presented here could be useful for further analysis of the establishment of the germ line in Drosophila.     

Clonal analysis of the tissue specificity of recessive female-sterile mutations of Drosophila melanogaster using a dominant female-sterile mutation Fs(1)K1237

Using the newly isolated, germ line-dependent dominant female-sterile mutation Fs(1)K1237, we have characterized the germ line or somatic line dependence of 25 X-linked recessive female-sterile mutations. Since Fs(1)K1237/+ females fail to lay eggs, only germ line cells which lose Fs(1)K1237 as a result of X-ray-induced mitotic recombination are capable of producing eggs. Such recombination events will render genes on the homologous chromosome homozygous. If this chromosome carries a recessive female-sterile mutation, the fertility will be restored only if the altered function is not required in the germ line. Using this test, we have classified 25 recessive female-sterile mutations: 12 affect germ line function, 12 affect somatic line function, and one gave an ambiguous result for which an explanation is proposed. For a few of the somatic line-dependent mutants, we found that some eggs derived from germ line clones showed the same phenotype as eggs laid by females homozygous for the recessive female-sterile mutation. These results are discussed in terms of a coincident production of clones in the follicle cells.     

Heterospecific combinations of germ cells and gonadal soma betweenDrosophila melanogaster,D. mauritiana andD. ananassae

Summary We transplanted pole cells betweenDrosophila melanogaster, D. mauritiana andD. ananassae to investigate the ability of germ cells to develop in the gonad of a heterospecific host, and to study the interaction between somatic follicle cells and the cells of the germ line in producing the species-specific chorion. FemaleD. mauritiana germ cells in aD. melanogaster ovary produced functional eggs with normal development potential. The same is true for the reciprocal combination. FemaleD. ananassae pole cells in aD. melanogaster host only developed to a very early stage and degenerated afterwards. None of the interspecific combinations of male pole cells led to functional sperm. We could not determine at what stage the transplanted male pole cells were arrested. The cooperation of follicle cells and the oocyte-nurse cell complex in producing the chorion was studied using the germ-line-dependent mutationfs(1) K10 ofD. melanogaster, which causes fused respiratory appendages and an abnormal chorion morphology. Wild-type femaleD. mauritiana germ cells in a mutantfs(1) K10 D. melanogaster ovary led to the production of wild-type eggs withD. melanogaster-specific, short respiratory appendages. On the other hand,D. melanogaster fs(1) K10 germ cells in aD. mauritiana ovary induced the formation of eggs with mutant fused appendages which were, however, typicallyD. mauritiana in length. When.D. mauritiana pole cells developed in aD. melanogaster ovary, the chorion exhibited a new imprint pattern that differs from both species-specific patterns.     

Two Drosophila learning mutants, dunce and rutabaga, provide evidence of a maternal role for cAMP on embryogenesis

The dunce gene of Drosophila melanogaster encodes a cAMP-specific phosphodiesterase (form II). Mutant dunce flies have elevated levels of cAMP and exhibit a number of defects including learning deficiencies and female sterility. Two partial suppressors of the female sterility phenotype have been selected in an X chromosome containing a dunce null mutation. Both suppressors are associated with reduced AC2 activity. Complementation analyses suggest that both are alleles of the learning mutant rutabaga. Females homozygous for dunce null mutations that abolish PDE activity do not deposit eggs. The suppressors exhibit differential effects on egg deposition and production of progeny; double-mutant females deposit many eggs that fail to hatch, but some develop to adults. These adult progeny exhibit morphological defects that are confined mostly to the second and third thoracic segments or to the first five abdominal segments. These observations demonstrate that the dunce gene is required in adult females for egg laying and that the dunce gene provides an essential maternal function required for normal development of the zygote. Clonal analysis, employing the dominant female-sterile mutation ovoD1, demonstrates that the former requirement for PDE activity resides in somatic cells and that the latter requirement resides in germ line cells. Female germ line cells homozygous for a dunce null mutation produce oocytes that fail to develop. Thus, homozygous dunce null-mutant zygotes develop to adults solely because of the enzyme or mRNA present in the oocytes of heterozygous mothers. Mutant alleles of rutabaga act in the germ line cells to partially suppress the developmental defects caused by dunce mutations. Thus the rutabaga gene, as well as the dunce gene, functions in both somatic and germ line cells.     

Normal Female Germ Cell Differentiation Requires the Female X Chromosome to Autosome Ratio and Expression of Sex-Lethal in DROSOPHILA MELANOGASTER

In somatic cells of Drosophila, the ratio of X chromosomes to autosomes (X:A ratio) determines sex and dosage compensation. The present paper addresses the question of whether germ cells also use the X:A ratio for sex determination and dosage compensation. Triploid female embryos were generated which, through the loss of an unstable ring-X chromosome, contained some germ cells of 2X;3A constitution in their ovaries. Such germ cells were shown to differentiate along one of two alternative pathways: a minority developed into normal female oocytes and eggs; the majority developed into abnormal multicellular cysts. An X:A ratio of 1 is, therefore, required in female germ cell development, at least in the mature ovary after stem cell division. Abnormal development of female germ cells was also observed when 2X;2A germ cells which were homozygous or trans-heterozygous for mutant alleles at the Sex-lethal locus were transplanted into normal female host embryos at the blastoderm stage. Germ cells homozygous for amorphic alleles failed to give rise to normal eggs. Instead, they formed multicellular cysts, very similar to those formed by 2X;3A cells. Zygotic Sxl+ activity is, therefore, also necessary for the development of normal female germ cells. No abnormalities were detected in transplanted germ cells from female embryos whose mothers had been homozygous for the mutation daughterless. When normal XY germ cells were transplanted into female embryos, no traces of such cells could be found in the adult ovary. XY germ cells seem, therefore, not to develop as far as 2X;3A or Sxl homozygous cells in a female gonad. This indicates that neither 2X;3A nor Sxl homozygous germ cells are equivalent to normal XY germ cells.     

Requirement for Cell-Proliferation Control Genes in Drosophila Oogenesis

Genes that are required for cell proliferation control in Drosophila imaginal discs were tested for function in the female germ-line and follicle cells. Chimeras and mosaics were produced in which developing oocytes and nurse cells were mutant at one of five imaginal disc overgrowth loci (fat, lgd, lgl, c43 and dco) while the enveloping follicle cells were normal. The chimeras were produced by transplantation of pole cells and the mosaics were produced by X-ray-induced mitotic recombination using the dominant female-sterile technique. The results show that each of the genes tested plays an essential role in the development or function of the female germ line. The fat, lgl and c43 homozygous germ-line clones fail to produce eggs, indicating a germ-line requirement for the corresponding genes. Perdurance of the fat+ gene product in mitotic recombination clones allows the formation of a few infertile eggs from fat homozygous germ-line cells. The lgd homozygous germ-line clones give rise to a few eggs with abnormal chorionic appendages, a defect thought to result from defective cell communication between the mutant germ-line and the nonmutant follicle cells. One allele of dco (dcole88) prevents egg development when homozygous in the germ line, whereas the dco18 allele has no effect on germ-line development. Fs(2)Ugra, a recently described follicle cell-dependent dominant female-sterile mutation, allowed the analysis of egg primordia in which fat, lgd or lgl homozygous mutant follicle cells surrounded normal oocytes. The results show that the fat and lgd genes are not required for follicle cell functions, while absence of lgl function in follicles prevents egg development.(ABSTRACT TRUNCATED AT 250 WORDS)     

The maternal and zygotic roles of the gene Polycomb in embryonic determination in Drosophila melanogaster

The mutation Polycomb (Pc) is known to cause a variety of intersegmental transformations in homozygous and heterozygous individuals of Drosophila melanogaster; Pc⁺ is thought to act as a negative regulator of genes of the bithorax complex. The function of this gene in the maternal germ line has been assessed by examining the variation in expression of these homoeotic phenotypes in individuals derived from a maternal germ line with a single or no dose of the Pc⁺ allele. Mosaic individuals with a homozygous or heterozygous Pc germ line were produced by transplantation of pole cells, the embryonic precursors of the germ line. By employing an X-linked dominant female-sterile mutation, the identification of mosaic females and the study of progeny derived from the exogenous germ line were greatly simplified; the advantages of this system for the transplantation of pole cells for such analyses are described. In general, all thoracic and abdominal segments of homozygous Pc embryos differentiate characteristics of the eighth, most posterior, abdominal segment. The extent and uniformity of this transformation as well as other manifestations of the homozygous Pc genotype are described and shown to be correlated with the maternal germ line genotype; homozygous Pc embryos derived from a homozygous Pc maternal germ line show greater expression of these phenotypes than do genetically identical embryos derived from a heterozygous Pc maternal germ line. The expression of some homoeotic phenotypes typical of heterozygous Pc adults shows only a slight correlation with the maternal genotype, while no homoeotic transformations are clearly evident in heterozygous larvae of either origin. Thus, the maternal effect of Pc is rescuable. The results suggest that the Pc⁺ gene is active in the maternal germ line but that the absence of the maternally derived Pc⁺ product can be largely compensated by the introduction of a wild-type allele upon fertilization; this rescue indicates that the maternal activity of Pc⁺ plays no major role in the normal process of embryonic segmental determination. The normal fertility of males and females with a homozygous Pc germ line and of their progeny suggests that Pc⁺ plays no role in the determination or development of the germ line in either the maternal or zygotic genome.     

Studies on the embryonic diapause of thepnd mutant of the silkworm,Bombyx mori

Summary The changing pattern in free amino acids following the embryonic development in the non-diapause and diapause eggs of thepnd mutant of theBombyx silkworm was studied. In the diapause eggs, heterozygous for thepnd gene, the levels of most of the amino acids increased concomitantly with the substantial decrease in oxygen consumption. Among the amino acids, alanine was the only amino acid that showed a large accumulation. The accumulation could be induced experimentally in the non-diapause eggs, homozygous for thepnd gene, by reducing the oxygen supply. In contrast, it was prevented in the diapause eggs by increasing the oxygen supply. From these results, it is suggested that the alanine accumulation is the consequence of anaerobic metabolism in the eggs during diapause. The possible significance of the alanine accumulation is discussed in relation to the anaerobic carbohydrate metabolism associated with the embryonic diapause in thepnd mutant.     

The PSO3 gene is involved in error-prone intragenic recombinational DNA repair in Saccharomyces cerevisiae

Summary The induction of gene conversion and mitotic crossing-over by photoaddition of psoralens, 254 nm ultraviolet radiation, and nitrogen mustards was determined in diploid cells homozygous for the pso3-1 mutation and in the corresponding wild type of Saccharomyces cerevisiae. For these different agents, the frequency of non-reciprocal events (conversion) is reduced in the pso3-1 mutant compared to the wild type. In contrast, the frequency of reciprocal events (crossing-over) is increased at a range of doses. These observations, together with the block in induced mutagenesis for both reverse and forward mutations previously reported for the pso3-1 mutant, suggest that the PS03 gene product plays a role in mismatch repair of short patch regions. The block in gene conversion in the pso3 homozygous diploid leads, in the case of nitrogen mustards, to specific repair intermediates which are lethal to the cells.     

The use of rootless mutants for the screening of spontaneous androgenetic and gynogenetic haploids in Nicotiana tabacum: evidence for the direct transfer of cytoplasm

Summary In crosses between a homozygous rootless mutant line of Nicotiana tabacum used as female and other Nicotiana tabacum lines, androgenetic haploids can be directly selected by their ability to form plantlets with a normal rooting system, whereas hybrid plants are killed few weeks after sowing. These androgenetic plants have the nucleus of the male parent into the cytoplasm of the female parent. In crosses where the homozygous rootless mutant line is used as a pollen donor, gynogenetic haploids can also be directly selected. Haploids can therefore be derived from male sterile plants using this approach. A generalization of this system for direct cytoplasm transfer and for the screening of spontaneous haploids in dicotyledons is proposed.     

Maternal effects of general and regional specificity on embryos of Drosophila melanogaster caused by dunce and rutabaga mutant combinations

Summary The developmental patterns of embryos produced by female germ line cells homozygous for null-enzyme mutations of dunce and for dunce in combination with each of three different rutabaga mutations are compared with the normal pattern. At least three discrete developmental defects at progressive stages following fertilization can be identified and correlated with the loss of adenylate cyclase activity caused by rutabaga mutations, suggesting that the defects are caused by elevated cyclic AMP levels in female germ line cells. The earliest defect occurs soon after fertilization and affects DNA replication and mitosis, prevents nuclear migration, and leads to large polyploid nuclei. A later defect prevents cleavage nuclei from migrating into, or dividing in, the posterior region of the egg. The last affects the developmental behavior or fate of blastoderm cells. Some of these defects mimic those produced by previously described maternal-effect mutations.     

Maternal effects of zygotic mutants affecting early neurogenesis inDrosophila

Summary The size of the neurogenic region ofDrosophila melanogaster is under the control of several genes of zygotic expression. Lack of function from any of those genes produces an increase of the size of the neurogenic region at the expense of the epidermal anlage. However, differences exist in the extent of neuralisation achieved by each of the genetic loci upon mutation. The present results show that in the case ofN andmam phenotype differences are due to different contributions of maternal gene expression. This could be shown by studying the phenotype which appeared in mutant embryos when the oocytes developed from homozygous mutant precursor cells. Clones of mutant cells were induced in the germ line of females heterozygous for the neurogenic mutationin trans over germ line dependent, dominant female sterile mutations. After removing maternal information the phenotype ofN andmam mutants became identical in both cases. Furthermore maternal information fromN ⁺ was found to be necessary for viability of the wildtype.     

Autosomal control of lampbrush-loop formation during spermatogenesis in Drosophila hydei by a gene also affecting somatic sex determination

Summary The Y chromosome of Drosophila hydei carries information that is necessary for the development of the spermatozoa. In primary spermatocytes Y chromosomal genes become active: five of the male fertility factors form giant lampbrush loops. Our prior work indicated interactions between the Y chromosomal genes and autosomal loci. It is of interest to identify loci regulating the activity of the Y chromosomal genes. We, therefore, screened a total of about 14,000 chromosomes (X, 2, 3 and 4) for mutations that interfere with the expression of the lampbrush loops. Two mutations with substantial effects on the loop morphology were recovered. One of them, a recessive male sterile mutation (ms (3) 5) on chromosome 3, is described in this paper. Its homozygous state results in a complete absence of all Y chromosomal lampbrush loops at 26° C; at 18° C the loops are formed. Temperature shifts with homozygous males indicate that the function early during the spermatogonial stage is crucial for the development of lampbrush loops in the primary spermatocyte. Meiosis is entirely absent in the male, but normal in females. Females homozygous for ms (3) 5 display a maternal effect, which reduces the viability and fertility of homozygous daughters and produces sons with signs of intersexuality. Linkage studies indicated that the effect on the male germ line and the maternal effects cannot be separated and may hence be induced by a single gene.     

Mutant fs(1) 1163 of Drosophila melanogaster alters yolk protein secretion from the fat body

Summary The mutant fs(1) 1163 of Drosophila melanogaster, which was isolated by Gans et al. (1975) is a recessive homozygous female sterile at 18°C and a dominant female — sterile at 29°C. We reported previously that there are reduced quantities of the largest of the three yolk polypeptides in Drosophila melanogaster in the haemolymph and eggs of this mutant at 29°C (Bownes and Hames 1978 a). In this paper we show that the yolk protein defect maps within approximately 2.5 recombination units of the female sterility at 21±2.5 map units on the X-chromosome. The temperature-sensitive period of the yolk protein defect is after emergence. In vitro labelling of fs(1) 1163 ovaries and fat bodies showed that they were able to synthesise yolk polypeptide 1. Interestingly, studies on the proteins present in the various tissues indicate that the fat body tends to accumulate all three yolk polypeptides in the mutant. This phenotype is partially co-dominant in that an effect is seen in heterozygotes as well as homozygotes and is enhanced by increased temperature. This mutant could therefore have a defect (a) in the structural gene for yolk polypeptide 1, (b) in the processing and secretion enzyme systems; (c) in the fat body or all tissues leading to altered secretion properties.Mutants like fs(1) 1163 which alter specific steps in vitellogenesis should be of value for analysing the genetic and biochemical control of the synthesis, transport and sequestering of the yolk polypeptides during oogenesis.