Genetic analysis of viable and lethalfused mutants ofDrosophila melanogaster

Summary Fused is a segmentation gene belonging to the segment-polarity class. Mutations at thefused locus are known to display pleiotropic effects, causing zygotically determined anomalies of ovaries and of some adult cuticular structures, and maternally determined embryonic segmentation defects. In order to determine the amorphic phenotype offused and to study the genetical basis of its pleiotropy, newfused alleles (18 viable and 11 lethal) were isolated. The phenotype of these mutants and of others already known are described, taking into account zygotic and maternal effects. The main results provided by this analysis are as follows. Firstly, allfused alleles show the whole complex fused phenotype, and a good correlation is observed between the strength of the wing and segmentation defects, suggesting that a single function is involved in both processes. Secondly, all embryonic and larval lethals carry deficiencies which allow us to localizefused between the 17C4 and 17D2 bands of the X-chromosome. Thirdly, the 24 viable and 2 pupal lethals examined behave as point mutants, as shown cytologically or by Southern blot analysis. However, only one of them, the pupal lethalfu ^mH63 was proven to carry a nullfused allele, since it displays in germ-line clones a strong maternal phenotype and a very low zygotic rescue, similar to those of the small deficiencyDf(1)fu ^z4. The phenotype of the amorphic mutant indicates that zygotic ezpression offused is required for normal metamorphosis, while maternal expression is necessary for a normal segmentation pattern, since a complete loss offused expression during oogenesis cannot be compensated zygotically.     

Maternal Piwi regulates primordial germ cell development to ensure the fertility of female progeny in Drosophila

In many animals, germline development is initiated by proteins and RNAs that are expressed maternally. PIWI proteins and their associated small noncoding PIWI-interacting RNAs (piRNAs), which guide PIWI to target RNAs by base-pairing, are among the maternal components deposited into the germline of the Drosophila early embryo. Piwi has been extensively studied in the adult ovary and testis, where it is required for transposon suppression, germline stem cell self-renewal, and fertility. Consequently, loss of Piwi in the adult ovary using piwi-null alleles or knockdown from early oogenesis results in complete sterility, limiting investigation into possible embryonic functions of maternal Piwi. In this study, we show that the maternal Piwi protein persists in the embryonic germline through gonad coalescence, suggesting that maternal Piwi can regulate germline development beyond early embryogenesis. Using a maternal knockdown strategy, we find that maternal Piwi is required for the fertility and normal gonad morphology of female, but not male, progeny. Following maternal piwi knockdown, transposons were mildly derepressed in the early embryo but were fully repressed in the ovaries of adult progeny. Furthermore, the maternal piRNA pool was diminished, reducing the capacity of the PIWI/piRNA complex to target zygotic genes during embryogenesis. Examination of embryonic germ cell proliferation and ovarian gene expression showed that the germline of female progeny was partially masculinized by maternal piwi knockdown. Our study reveals a novel role for maternal Piwi in the germline development of female progeny and suggests that the PIWI/piRNA pathway is involved in germline sex determination in Drosophila.     

The effects of two mutations connected with chromatin functions on female germ-line cells of Drosophila

Summary We have studied the developmental effects of two dominant suppressor mutations of position-effect variegation mutations on female germ-line cells. Su-var (2) 1⁰¹, which has been shown to affect chromatin structure though altering histone deacetylation, and Su-var (3) 3⁰³are recessive female steriles and zygotic lethals in the presence of butyrate or an additional Y chromosome. We have analysed mosaic females with mutant germ-line and normal soma and concluded that intact functions of the Su-var (2) 1 and the Su-var (3) 3 genes are required for development of both the soma and the germ-line and that as indirect evidence suggest, their maternally provided products are needed for normal embryonic development. It is suggested that there is possibly a common control of chromatin structure and gene expression in the soma, female germ-line and embryonic cells of Drosophila.     

Epigenetic Regulation of Programmed Genomic Rearrangements in Paramecium aurelia1

ABSTRACT In ciliates, development of the polyploid somatic macronucleus after sexual events involves extensive and reproducible rearrangements of the germ-line genome, including chromosome fragmentation and precise excision of numerous internal sequence elements. In Paramecium aurelia, alternative macronuclear versions of the same germ-line genome can be maternally inherited across sexual generations, showing that rearrangement patterns are not strictly determined by the germ-line sequence. Homology-dependent maternal effects can be evidenced by transformation of the vegetative macronucleus with cloned macronuclear sequences: new fragmentation patterns or internal deletions are specifically induced during differentiation of a new macronucleus, in sexual progeny of transformed clones. Furthermore, transformation of the maternal macronucleus with germ-line sequences containing internal eliminated sequences (short single-copy elements) can result in a specific inhibition of the excision of the same elements in the zygotic macronucleus. These experiments show that the processing of many germ-line sequences in the developing macronucleus is sensitive to the structure and copy number of homologous sequences in the maternal macronucleus. The generality and sequence specificity of this trans-nuclear, epigenetic regulation of rearrangements suggest that it is mediated by pairing interactions between germ-line sequences and sequences imported from the maternal macronucleus.     

Viability of Female Germ-Line Cells Homozygous for Zygotic Lethals in DROSOPHILA MELANOGASTER

We have analyzed the viability of different types of X chromosomes in homozygous clones of female germ cells. The chromosomes carried viable mutations, single-cistron zygotic-lethal and semi-lethal mutations, or small (about six chromosome band) deletions. Homozygous germ-line clones were produced by recombination in females heterozygous for an X-linked, dominant, agametic female sterile.

All the zygotic-viable mutants are also viable in germ cells. Of 16 deletions tested (uncovering a total of 93 bands) only 2 (of 4 and 5 bands) are germ-cell viable. Mutations in 15 lethal complementation groups in the zeste-white region were tested. When known, the most extreme alleles at each locus were tested. Only in five loci (33%) were the mutants viable in the germ line. Similar studies of the same deletions and point-mutant lethals in epidermal cells show that 42% of the bands and 77% of the lethal alleles are viable. Thus, germ-line cells have more stringent cell-autonomous genetic requirements than do epidermal cells.

The eggs recovered from clones of three of the germ-cell viable zw mutations gave embryos arrested early in embryogenesis, although genotypically identical embryos derived from heterozygous oogonia die as larvae or even hatch as adult escapers. For two genes, homozygosis of the mutations tested also caused embryonic arrest of heterozygous female embryos, and in one case, the eggs did not develop at all. Germ-line clones of one quite leaky mutation gave eggs that were indistinguishable from normal. The abundance of genes whose products are required for oogenesis, whose products are required in the oocyte, and whose activity is required during zygotic development is discussed.

     

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.     

JAK-STAT signalling is required throughout telotrophic oogenesis and short-germ embryogenesis of the beetle Tribolium

In Drosophila, the JAK-STAT signalling pathway regulates a broad array of developmental functions including segmentation and oogenesis. Here we analysed the functions of Tribolium JAK-STAT signalling factors and of Suppressor Of Cytokine Signalling (SOCS) orthologues, which are known to function as negative regulators of JAK-STAT signalling, during telotrophic oogenesis and short-germ embryogenesis. The beetle Tribolium features telotrophic ovaries, which differ fundamentally from the polytrophic ovary of Drosophila. While we found the requirement for JAK-STAT signalling in specifying the interfollicular stalk to be principally conserved, we demonstrate that these genes also have early and presumably telotrophic specific functions. Moreover, we show that the SOCS genes crucially contribute to telotrophic Tribolium oogenesis, as their inactivation by RNAi results in compound follicles. During short-germ embryogenesis, JAK-STAT signalling is required in the maintenance of segment primordia, indicating that this signalling cascade acts in the framework of the segment-polarity network. In addition, we demonstrate that JAK-STAT signalling crucially contributes to early anterior patterning. We posit that this signalling cascade is involved in achieving accurate levels of expression of individual pair-rule and gap gene domains in early embryonic patterning.     

Expression patterns of twist and snail in Tribolium (Coleoptera) suggest a homologous formation of mesoderm in long and short germ band insects

The mesodermal region in Drosophila is determined by a maternally derived morphogenetic gradient system which specifies the different cell fates along the dorsoventral axis, including the prospective mesodermal cells at the ventral side of the embryo. There are at least two zygotic target genes, twist and snail, which are required for mesoderm formation in Drosophila. To analyze whether a similar mode of mesoderm specification might also apply to short germ band insect embryos, we have cloned twist and snail- related gene fragments from the flour beetle Tri-bolium and have analyzed their expression pattern. Both genes are expressed in a ventral stripe at early blastoderm stage, which is restricted to the region of the developing germ rudiment. The cells expressing the two genes are those that invaginate during gastrulation, indicating that the early stages of mesoderm specification are indeed very similar between the two species. Interestingly, both genes are also expressed during germband extension in a subregion of the growth zone of the embryo which forms the mesodermal cells. This suggests that the expression of the two genes is required for mesoderm formation both at early blastoderm stage and during germband elongation until the end of the segmental growth process. © 1994 Wiley-Liss, Inc.     

Developmental analysis of fs(1)gastrulation defective,a dorsal-group gene of Drosophila melanogaster

Summary The gastrulation defective (gd) locus is a maternally expressed gene in Drosophila required for normal differentiation of structures along the embryonic dorso-ventral axis. Cuticular defects of the offspring from females with different combinations of gd alleles comprised a phenotypic continuum. Complementation among several alleles produced normal offspring while progressively more severe mutations produced a graded loss of structures from ventral, and then lateral, blastoderm cells. The most severely affected embryos consisted entirely of structures derived from dorsal blastoderm cells. Histological examination of staged siblings from selected allelic combinations showed that internal tissues were similarly affected. The tissues observed in amorphic embryos support new, more dorsal, assignments of fate map positions for blastoderm precursors of the cephalopharyngeal apparatus, hindgut and ventral nerve cord. The loss of ventral and lateral structures did not occur through cell death and appeared to involve a change in blastoderm cell fate. A direct effect of the mutations on blastoderm cell determination, however, was insufficient to explain the development of the dorsalized embryos. Intermediate phenotypes suggested that cell interactions or movements associated with morphogenesis are required for the determination of some cell fates in the dorsoventral axis. Thus, the developmental fate of all blastoderm cells may not be fixed at the time of blastoderm formation.     

Maternal Nanos and Pumilio regulate zygotic vasa expression autonomously in the germ-line progenitors of Drosophila melanogaster embryos

vasa (vas) is transcribed earliest among reported genes expressed in the germ-line progenitors, or pole cells, in Drosophila melanogaster embryos. Its expression is detected in the germ-line cells throughout their development, making vas expression a useful marker for the establishment of germ-line fate. In the present report, it is shown that maternal Nos and Pum are required for normal expression of vas in pole cells. First, expression of enhancer-trap marker BC69, which reflects vas expression, is promoted by maternal Nos and Pum. Second, expression of vas mRNA in pole cells is promoted by maternal Nos and Pum. Third, pole cell transplantation experiments reveal that maternal Nos and Pum are required autonomously in pole cells for proper expression of vas. Finally, Nos and Pum are dispensable for vas expression in oogenesis, although they are expressed zygotically in adult ovaries. These observations show that germ-line-specific vas expression is promoted by autonomous function of maternal Nos and Pum in the germ-line progenitors during embryogenesis, and is regulated differentially in embryogenesis and oogenesis.     

The effects of zygotic lethal mutations on female germ-line functions in Drosophila

Many genetic loci that result in lethality when mutated may also have an essential role in oogenesis. The maternal effects of EMS-induced zygotic lethal mutations at 48 loci were examined using the dominant female-sterile technique. Three categories of effects were found. In the first group (13 out of 48), no maternal effect was detected. The second set (20 out of 48) exhibited maternal effects on oogenesis, embryogenesis, or both. In 13 of this last group, only a few eggs were produced before a progressive deterioration of development occurred. It is suggested that perdurance of the wild-type gene product could produce this result. The third group (15 out of 48) produced cell lethality in germ-line clones, an effect that may be related to their role in indispensable cell functions. Three loci were found which, in germ-line clones, produced embryonic phenotypes that resemble maternal effect mutations. The implications of this study for the genetic analysis of early development are discussed.     

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.     

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.