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.     

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.     

A pupal lethal mutation with a paternally influenced maternal effect on embryonic development in Drosophila melanogaster

The maternal effect and zygotic phenotype of l(1)pole hole (l(1)ph) is described. l(1)ph is a zygotic lethal mutation which affects cell division of adult precursor cells in Drosophila larvae. The locus is located in 2F6 on the salivary gland chromosome map and four alleles have been characterized. Germ-line clonal analysis of amorphic alleles indicates that l(1)ph has a maternal effect lethal phenotype. Two lethal phenotypes are observed among embryos derived from female germ-line clones homozygous for amorphic alleles dependent upon the zygotic activity of l(1)ph⁺ introduced via the sperm. Class 1: If no wild-type dose of the gene is introduced, embryos form abnormal blastoderms in which nuclear migration and cell formation is disrupted leading to an ill-defined cuticular pattern. Class 2: If a wild-type copy of the gene is introduced, blastoderm cells do not form beneath the pole cells (the pole hole phenotype); subsequently such embryos are missing cuticular structures posterior to the seventh abdominal segment (the torso phenotype). When the zygotic activity l(1)ph⁺ is modulated using position effect variegation a new phenotype is observed among class 2 embryos in which torso embryos are twisted along their longitudinal axis.     

Analysis of sex determination in the monogenic blowfly Chrysomya rufifacies by pole cell transplantation

Summary Sex determination in the monogenic blowfly Chrysomya rufifacies is controlled by a dominant or epistatic female sex realizer (F) having sex predetermining properties (F/f=female-producing female; f/f=male-producing female or male, respectively). To determine (1) the cell type in which the maternal effect gene F is expressed. and (2) the autonomous or nonautonomous sexual differentiation of the germ cells germ-line mosaics were constructed in C. rufifacies by pole-cell transplantations. The production of bisexual progeny by germ-line mosaics generated by transplanting pole cells between both types of female embryos shows that the F gene product is synthesized by germ-line cells themselves, not by maternal (intra- or extraovarian) somatic cells. Pole cell transplantations between male and female embryos yielded completely fertile heterosexual germ-line mosaics thus demonstrating phenotypic sex reversal of donor germ cells in a host of the opposite sex. Consequently, the sexual differentiation of a germ cell in C. rufifacies is not determined by its own genotypic constitution but is induced by the surrounding somatic cells.The male sex of F/f individuals generated by fertilization with F-bearing sperm from a heterosexual germ-line mosaic indicates that the F gene is either not expressed during spermatogenesis and early embryogenesis or is expressed too late or in not sufficient amounts to direct differentiation into the female sex. This finding is consistent with the assumption that progamic expression of F is found exclusively during oogenesis in F/f females.     

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.     

The development and function of the female germ line in Drosophila melanogaster: a cell lineage study.

X-ray-induced mitotic recombination was used to follow the development and function of the female germ line in Drosophila melanogaster. Clones marked by maternal effect mutations which alter the morphology of the egg [fs(1)K10] or the phenotype of the resulting progeny (maroonlike) were produced in trans-heterozygotes irradiated during embryonic, larval, or pupal development or as 5-day-old adults. Judging from the size of clones induced at the blastoderm stage, only five to ten of the pole cells observed on the surface of the embryo contribute to the germ line. Most of the K10 clones induced during embryonic and larval development were associated with mal twin spots, indicating that both daughters of the irradiated germ cell remained in the germ line and gave rise to eggs in the adult. During larval life the number of cells increases logarithmically and reaches a maximum of 110 at 24 hr after pupation. The same value was obtained for 5-day-old adults. In contrast to the mosaic females produced as embryos and larvae, mosaics obtained after pupal and adult irradiations were of two types, those laying only one K10 egg and those laying several K10 eggs distributed over the lifespan of the adult. This result indicates that the stem cell divisions characteristic of the adult period have begun shortly after pupation. About 9 to 11 days are required for an irradiated stem cell to produce its first clonal K10 egg, and two-thirds of this time is spent in the germarium. Each ovariole possesses on the average two to three functioning stem cells. This multiplicity of stem cells was confirmed by the recovery of mosaic ovarioles when mal heterozygotes irradiated as adults or late larvae were stained for aldehyde oxidase activity.     

Injection of wild-type cytoplasm and poly(A)+ RNA provokes phenotype rescue in spätzle mutant Drosophila embryos

Summary spätzle (spz), a maternal effect gene of Drosophila, is involved in the establishment of the dorso-ventral axis during embryogenesis. Eggs from females lacking the spz gene product develop into completely dorsalized embryos, i.e. the ventral and lateral pattern elements fail to develop. Upon injection of either cytoplasm or poly(A)⁺ RNA from early wild-type embryos, spz embryos develop lateral pattern elements represented by Filzkörper and in the case of injected cytoplasm additional ventral pattern elements represented by ventral setae. Wild-type cytoplasm retains the rescuing activity longer than the poly(A)⁺ RNA fraction does, and cytoplasm is always more effective in provoking the rescue than poly(A)⁺ RNA. Mosaic females containing spz germ cells surrounded by spz ⁺ tissues were generated by pole cell transplantations; a mutant genotype in the germ cells is sufficient to produce all aspects of the spz mutant phenotype, suggesting that the maternal source of spz gene product is the germ line.     

Expression of xanthine dehydrogenase activity during embryonic development of Drosophila melanogaster

The contributions of oogenesis and zygotic genome expression to xanthine dehydrogenase activity during embryogenesis were investigated utilizing the mal and ry² mutants. In vitro complementation experiments demonstrated the presence of the mal⁺ complementation factor in the oocyte, suggesting an explanation for the mal maternal effect. The ry⁺ complementation factor synthesized from paternal template was detected at gastrulation. This is the earliest detection of a paternal enzyme during nonmammalian embryonic development.     

A method for studying tissue specificity of maternally affected Drosophila melanogaster mutants: mosaic analysis of cinnamon.

Mosaic analysis is introduced to study tissue specificity of the maternal effect which characterizes the wild-type allele of cinnamon (=cin). The methodology presented is applicable to studies of other maternal effect mutations, as well as to female or male sterility mutations. One hundred and forty-three pal-induced fertile cin-mosaic females were obtained, and the degree to which they were capable of maternally affecting their homozygous cin daughters was correlated to their cuticular and germinal genetic constitution. From this analysis the following conclusions are drawn: (1) The presence of a wild-type (cin⁺) allele in maternal germ cells constitutes a sufficient condition for the full expression of the cin⁺ characteristic maternal effect: All cin offspring derived from such cells have normal viability and eye color. (2) This effect is confined to the descendants of a particular germ cell and does not extend to the descendants of other non-cin⁺ germ cells in the same or in a neighboring ovary. (3) A wild-type allele in a germ cell constitutes a necessary condition for the eye color maternal effect. (4) When a maternal germ line is wholly mutant, nonmutant constitution of an additional focus may result in rescue of more than half of the homozygous cin offspring (all with mutant eye color). Mosaic analysis suggests that this somatic viability focus originates from the posterior region of the blastoderm. These conclusions were tested and confirmed by transplanting heterozygous cin ovaries, wild-type Malphigian tubes, and wild-type fat body into homozygous cin hosts. In addition, transplantations of homozygous cin ovaries into wild-type hosts suggest that the posterior maternal viability focus corresponds to the mesodermal components of the ovaries.     

Autonomy of the wingless mutation in Drosophila melanogaster

Summary T(Y;2) translocations were used to cytologically localise the wingless locus of Drosophila melanogaster. We found that an existing T(Y;2), which is an insertion of a segment of 2L into the Y chromosome, has wg ⁺ within this insert. This Y chromosome was used to generate an attached XY chromosome containing wg ⁺. The mutation claret-nondisjunctional (ca ^nd) was used to induce the loss of this XY chromosome and thus generate gynandromorphs with wg ¹/wg ¹ male tissue and wg ⁺/wg ¹/wg ¹ female tissue. Analysis of these gynanders demonstrated that a genotypically wingless mutant hemithorax is usually also phenotypically mutant in these half body mosaics; thus wg ¹ is discautonomous. This observation is of interest as it is known that wg is not cell autonomous.     

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.     

Temperature sensitivity of complementation at the Maroonlike eye color locus in Drosophila melanogaster

Summary In contrast to the wild-type eye color seen when Drosophila melanogaster heterozygous for mal ¹/mal ^F1 are cultured at 25 C, a mutant eye color is observed in heterozygotes cultured at 29–30 C throughout development. Furthermore, heterozygotes have wild-type eyes upon emergence provided development proceeds at 25 C during either of two critical periods: 1) The third quarter of the 3rd larval instar or 2) an imprecisely defined pupal period beginning less than 12 hours before the visual appearance of brown eye pigment and terminating about the time pigment appears in the wings.     

The isolation of functional pole cells from theDrosophila melanogaster maternal effect mutantmat(3)1

Summary A procedure for pole cell isolation has been developed that takes advantage of theDrosophila melanogaster maternal effect mutantmat(3) 1. Embryos derived from homozygousmat(3)1 mothers form exclusively pole cells. By outcrossing we could substantially increase the expressivity of the original mutant stock. We further introduced theTM8 balancer chromosome, which carries the dominant temperature sensitive mutationDTS-4. This allows the accumulation of large homozygousmat(3) 1 fly populations by eliminating the heterozygous flies at the restrictive temperature.Early embryos were mechanically fragmented and the cells were isolated by means of metrizamide step gradients. The isolated cells were demonstrated to exhibit the various ultrastructural and histochemical characteristics of pole cells. The isolated cells were transplanted into genetically marked host embryos. The germ line mosaics that were obtained indicate that the isolated cells represent functional pole cells.Proteins synthesized by the isolated pole cells during short term in vitro labelling with³⁵S-methionine were compared to the proteins synthesized by blastoderm cells fromOregon-R embryos. At least one protein could be demonstrated in the pole cell samples that is not synthesized byOregon-R blastoderm cells.The method allows a fast and gentle isolation of highly enriched pole cell populations which are a prerequisite for the biochemical analysis of germ cell determination and differentiation.     

Molecular analysis of a cytoplasmic factor essential for pole cell formation in Drosophila embryos

This article reviews recent analytical studies of cytoplasmic factors involved in a mechanism underlying pole cell formation in Drosophila embryogenesis. Polar plasm, or germ plasm, includes sources of two independent functions in the germ-line segregation from the somatic line: pole cell formation and commitment of pole cells to germ cells. The UV-caused inability of pole cell formation in embryos was restored by poly(A)⁺ RNA, of which cDNA was cloned. The nucleotide sequence of the cDNA was highly homologous with mitochondrial large rRNA.     

Difference in protein synthesis of ovaries indicates predetermined sex inChrysomya rufifacies (Diptera,Calliphoridae)

Summary Sex in the monogenic blowflyChrysomya rufifacies is under the control of a germ-line autonomous maternal effect sex realizer. In order to identify the ovarian poly(A)⁺RNA that might be related to sex predetermination, we analysed the patterns of cell-free translation products of total poly(A)⁺RNA from female and male predetermined ovaries and oocytes. During vitellogenesis we observed one transient sex-linked difference in a stable pattern composed of more than 800 in vitro translated proteins. This difference, however, was no longer detectable in mature oocytes.     

Germ-line cysts are formed during oogenesis in Erpobdella octoculata (Annelida,Clitellata, Erpobdellidae)

Abstract

Erpobdella octoculata (Clitellata, Hirudinea, Erpobdellidae) has paired ovarian sacs, each containing several rod-shaped structures termed ovarian bodies. Oogenesis takes place within the ovarian bodies. We show that in the apical part of the bodies the germ-line cells form syncytial cysts of cells interconnected by stable intercellular bridges. Germ-line cyst architecture is broadly similar to that of other clitellate annelids; that is, each germ cell has only one intercellular bridge connecting it to the anuclear cytoplasmic mass, the cytophore. Unlike germ-line cysts described in other leech species, the cytophore in cysts of E. octoculata is poorly developed, taking the form of thin cytoplasmic strands. Oogenesis in E. octoculata is meroistic because the germ cells forming the cysts (cystocytes) have diverse fates, i.e., nurse cells and oocytes appear. One large ramified cell (apical cell) occurs within the apical part of the ovarian body. We compare the ultrastructure of the apical cell found in E. octoculata with that of apical cells described recently in some hirudiniform leeches. The germ-line cysts as well as the oocytes are enveloped by somatic follicular cells. As in other leeches, the follicular cells surrounding the growing oocytes have cytoplasm perforated by intracellular canals. In view of the many similarities between E. octoculata ovarian bodies and the ovary cords described in glossiphoniids and especially in hirudiniform leeches, we suggest that the ovarian bodies found in E. octoculata are in fact modified ovary cords.     

The site of function of the Y chromosome in Drosophila melanogaster males

Summary The Y chromosome is essential for fertility in D. melanogaster males. An analysis of 126 pal-induced Y chromosome mosaics indicated that its function is only required in the germ line of fertile males. This analysis also showed that approximately 1/4 of all pal-induced Y chromosome mosaics had an XO/XYY constitution and hence that they resulted from somatic nondisjunction. Preliminary evidence suggests that pal-induced somatic nondisjunction can occur at the second or subsequent cleavage divisions.     

Germ line dependence of the deep orange maternal effect in Drosophila

Pole cell transplantations were used to determine the tissue specificity of maternal effects in Drosophila. The deep orange maternal effect is shown to be germ line autonomous. A cytoplasmic injection assay was used to determine when the dor⁺ substance could be detected in the developing oocyte. The dor⁺ substance is present during the early stages of vitellogenesis but could not be detected in the yolk of the embryo after blastoderm cellularization.     

Characterization of a region of the X chromosome of Drosophila including multi sex combs (mxc), a Polycomb group gene which also functions as a tumour suppressor

Genetic analysis of the 8D3;8D8-9 segment of the Drosophila melanogaster X chromosome has assigned seven complementation groups to this region, three of which are new. A Polycomb group (Pc-G) gene, multi sex combs (mxc), is characterized and mutant alleles are described. Besides common homeotic transformations characteristic of Pc-G mutants that mimic the ectopic gain of function of BX-C and ANT-C genes, mxc mutants show other phenotypes: they zygotically mimic, in males and females, the characteristic lack of germ line seen in progeny of some maternal effect mutants of the so-called posterior group (the grandchildless phenotype). Loss of normal mxc function can promote uncontrolled malignant growth which indicates a possible relationship between Pc-G genes and tumour suppressor genes. We propose that gain-of-function of genes normally repressed by the wild-type mxc product could, in mxc mutants, give rise to an incoherent signal which would be devoid of meaning in normal development. Such a signal could divert somatic and germ line developmental pathways, provoke the loss of cell affinities, but allow or promote growth.     

Altered SOS induction associated with mutations in recF, recO and recR

The SOS system of Escherichia coli aids survival following damage to DNA by promoting DNA repair while cell division is delayed. Induction of the SOS response is dependent on RecA and also on the product of recF. We show that normal induction also requires the products of recO and recR. SOS induction was monitored using a sfiA-lacZ fusion strain. Induction was delayed to a similar degree by mutation in recF, recO or recR. A similar effect was observed following overexpression of RecR from a recombinant recR ⁺plasmid. We show that the overexpression of RecR also reduces the UV resistance of a recBC sbcBC strain and of a sfiA strain, but not of a rec ⁺ sfiA ⁺strain. The implications of these data for the kinetics of DNA repair are discussed.