Control of mating plug expelling and sperm storage in Drosophila: A gynandromorph- and mutation-based dissection.

INTRODUCTION: In this study, we analyzed gynandromorphs with female terminalia, to dissect mating-related female behaviors in Drosophila. MATERIALS AND METHODS: We used gynandromorphs, experimentally modified wild-type (Oregon-R) females, and mutant females that lacked different components of the female reproductive apparatus. RESULTS: Many of the gynandromorphs mated but did not expel the mating plug (MP). Some of these - with thousands of sperm in the uterus - failed to take up sperm into the storage organs. There were gynandromorphs that stored plenty of sperm but failed to release them to fertilize eggs. Expelling the MP, sperm uptake into the storage organs, and the release of stored sperm along egg production are separate steps occurring during Drosophila female fertility. Cuticle landmarks of the gynandromorphs revealed that while the nerve foci that control MP expelling and also those that control sperm uptake reside in the abdominal, the sperm release foci derive from the thoracic region of the blastoderm. DISCUSSION AND CONCLUSION: The gynandromorph study is confirmed by analyses of (a) mutations that cause female sterility: Fs(3)Avar (preventing egg deposition), Tm2gs (removing germline cells), and iab-4DB (eliminating gonad formation) and (b) by experimentally manipulated wild-type females: decapitated or cut through ventral nerve cord.

[Pre-programmed genes]. / Eloprogramozott gének.

Cells feel good and carry on perfect functions when they contain the right types of proteins in the right concentration, at the right time and sites. There are mechanisms that ensure the right level of gene expression in the different cell types: the formation of protein molecules based on the DNA-encoded genetic information. Gene expression can also be regulated through the compactness of chromatin, i.e. the accessibility of the genes. The chromosomes are repositories of the genetic information - the sequence of base pairs - and also of the so-called epigenetic mechanisms that control gene expression through the regulation of chromatin compactness. The epigenetic mechanisms operate through DNA methylation and/or the regulation of chromatin compactness. The present overview takes a look into the phenomenon of epigenesis. It summarizes how genetic crosses reveal the involvement of epigenesis, explains its meaning and impact on life of the organisms. An understanding of epigenesis provides guidance to improve our life. Orv Hetil. 2017; 158(34): 1323-1330.

Towards long term cultivation of Drosophila wing imaginal discs in vitro.

The wing imaginal disc of Drosophila melanogaster is a prominent experimental system for research on control of cell growth, proliferation and death, as well as on pattern formation and morphogenesis during organogenesis. The precise genetic methodology applicable in this system has facilitated conceptual advances of fundamental importance for developmental biology. Experimental accessibility and versatility would gain further if long term development of wing imaginal discs could be studied also in vitro. For example, culture systems would allow live imaging with maximal temporal and spatial resolution. However, as clearly demonstrated here, standard culture methods result in a rapid cell proliferation arrest within hours of cultivation of dissected wing imaginal discs. Analysis with established markers for cells in S- and M phase, as well as with RGB cell cycle tracker, a novel reporter transgene, revealed that in vitro cultivation interferes with cell cycle progression throughout interphase and not just exclusively during G1. Moreover, quantification of EGFP expression from an inducible transgene revealed rapid adverse effects of disc culture on basic cellular functions beyond cell cycle progression. Disc transplantation experiments confirmed that these detrimental consequences do not reflect fatal damage of imaginal discs during isolation, arguing clearly for a medium insufficiency. Alternative culture media were evaluated, including hemolymph, which surrounds imaginal discs during growth in situ. But isolated larval hemolymph was found to be even less adequate than current culture media, presumably as a result of conversion processes during hemolymph isolation or disc culture. The significance of prominent growth-regulating pathways during disc culture was analyzed, as well as effects of insulin and disc co-culture with larval tissues as potential sources of endocrine factors. Based on our analyses, we developed a culture protocol that prolongs cell proliferation in cultured discs.

Assaying benzene, a parquet varnish, and a synthetic thinner with respect to induction of in vivo chromosome loss in wing primordial cells of Drosophila.

An assay detecting the in vivo loss of mwh(+)Y, a genetically engineered Y chromosome, in cells of the Drosophila wing primordia was published recently. Loss of the mwh(+)Y chromosome in any of the wing-disk cells - in a multiple wing hairs homozygous background - leads to the formation of an mwh mosaic spot (clone) in the emerging wing. The frequency and the size of the mwh clones allow detection and quantitative evaluation of environmental and/or genetic agents inducing chromosome loss. Using this novel technique, we analyzed the potential of vapors of benzene, a parquet varnish, and a synthetic thinner to induce chromosome loss. Exposure to 0.047µg/ml benzene vapor for one day or to 0.175µg/ml for four hours resulted in a significantly elevated mwh clone-frequency confirming the ability of benzene to induce chromosome loss. A one-day exposure to vapors of a parquet varnish or a 6-h exposure to vapors of a synthetic thinner slightly, yet significantly elevated the frequency of chromosome loss. Results of the present paper show the potential of vapors of the analyzed parquet varnish and synthetic thinner to induce chromosome loss, and illustrate the usefulness of the new technique.

Viability, longevity, and egg production of Drosophila melanogaster are regulated by the miR-282 microRNA.

The first microRNAs were discovered some 20 years ago, but only a small fraction of the microRNA-encoding genes have been described in detail yet. Here we report the molecular analysis of a computationally predicted Drosophila melanogaster microRNA gene, mir-282. We show that the mir-282 gene is the source of a 4.9-kb-long primary transcript with a 5' cap and a 3'-poly(A) sequence and a mature microRNA of ∼25 bp. Our data strongly suggest the existence of an independent mir-282 gene conserved in holometabolic insects. We give evidence that the mir-282 locus encodes a functional transcript that influences viability, longevity, and egg production in Drosophila. We identify the nervous system-specific adenylate cyclase (rutabaga) as a target of miR-282 and assume that one of the main functions of mir-282 is the regulation of adenylate cyclase activity in the nervous system during metamorphosis.

'Poking' microtubules bring about nuclear wriggling to position nuclei.

Nuclei wriggle in the cells of the follicle epithelium of the Drosophila pre-vitellogenic egg primordia. Although similar phenomena have been reported for a number of cultured cell types and some neurons in the zebrafish embryo, the mechanism and importance of the process have remained unexplained. Wriggling involves successive sudden and random minor turns of the nuclei, approximately three twists per minute with roughly 12° per twist, one of which lasts typically for 14 seconds. Wriggling is generated by the growing microtubules seeded throughout the cell cortex, which, while poking the nuclei, buckle and exert 5-40 piconewtons over ∼16 seconds. While wriggling, the nuclei drift ∼5 µm in a day in the immensely growing follicle cells along the apical-basal axis from the apical to the basal cell region. A >2-fold excess of the microtubules nucleated in the apical cell region, as compared with those seeded in the basal cell cortex, makes the nuclei drift along the apical-basal axis. Nuclear wriggling and positioning appear to be tightly related processes: they cease simultaneously when the nuclei become anchored by the actin cytoskeleton; moreover, colchicine or taxol treatment eliminates both nuclear wriggling and positioning. We propose that the wriggling nuclei reveal a thus far undescribed nuclear positioning mechanism.

An assay to detect in vivo Y chromosome loss in Drosophila wing disc cells.

Loss of the Y chromosome in Drosophila has no impact on cell viability and therefore allows us to assay the impact of environmental agents and genetic alterations on chromosomal loss. To detect in vivo chromosome loss in cells of the developing Drosophila wing primordia, we first engineered a Y chromosome with an attP docking site. By making use of the ΦC31 integrase system, we site-specifically integrated a genomic transgene encompassing the multiple wing hair (mwh) locus into this attP site, leading to a mwh(+)Y chromosome. This chromosome fully rescues the mwh mutant phenotype, an excellent recessive wing cell marker mutation. Loss of this mwh(+)Y chromosome in wing primordial cells then leads to manifestation of the mwh mutant phenotype in mwh-homozygous cells. The forming mwh clones permit us to quantify the effect of agents and genetic alterations by assaying frequency and size of the mwh mosaic spots. To illustrate the use of the mwh(+)Y loss system, the effects of four known mutagens (X-rays, colchicine, ethyl methanesulfonate, and formaldehyde) and two genetic conditions (loss- and gain-of-function lodestar mutant alleles) are documented. The procedure is simple, sensitive, and inexpensive.

The involvement of Importin-ß and peroxiredoxin-6005 in mitochondrial biogenesis.

Importin-ß is encoded by the Ketel gene in Drosophila. Upon running out of the maternal Importin-ß dowry larvae without the Ketel gene slow down and before dying possess symptoms characteristic for mitochondrial cytopathies. Death of the larvae is almost certainly the consequence of ceasing import of proteins, including some of the transcription factors, into the nuclei. We report here that the ensuing altered gene expression pattern leads to cessation of mitochondrial biogenesis. A transcriptome comparison between larvae with and without Ketel gene revealed altered expression level for 30 genes that are all nuclear. The seven downregulated genes have C/EBP transcription factor binding site in their promoter. RNAi silencing the function of peroxiredoxin-6005, one of the 23 upregulated genes, leads to excessive mitochondrial biogenesis, free radical production and death of the larvae. It appears that peroxiredoxin-6005 is engaged in mitochondrial biogenesis possibly as a component of redox-signaling.

Putting together rather than taking apart.

The town of Ascona in Switzerland, nestled on the northern shore of Lago Maggiore, hosted the 112 participants in the first systems biology meeting focused on developmental biology. The EMBO workshop was held between 16 and 20 August and brought together a multidisciplinary group of scientists who use systems approaches to understand how the size and shape of multicellular organisms and organs are determined.

Nest desertion is not predicted by cuckoldry in the Eurasian penduline tit.

Engagement in extra-pair copulations is an example of the abundant conflicting interests between males and females over reproduction. Potential benefits for females and the risk of cuckoldry for males are expected to have important implications on the evolution of parental care. However, whether parents adjust parental care in response to parentage remains unclear. In Eurasian penduline tits Remiz pendulinus, which are small polygamous songbirds, parental care is carried out either by the male or by the female. In addition, one third of clutches is deserted by both male and female. Desertion takes place during the egg-laying phase. Using genotypes of nine microsatellite loci of 443 offspring and 211 adults, we test whether extra-pair paternity predicts parental care. We expect males to be more likely to desert cuckolded broods, whereas we expect females, if they obtain benefits from having multiple sires, to be more likely to care for broods with multiple paternity. Our results suggest that parental care is not adjusted to parentage on an ecological timescale. Furthermore, we found that male attractiveness does not predict cuckoldry, and we found no evidence for indirect benefits for females (i.e., increased growth rates or heterozygosity of extra-pair offspring). We argue that male Eurasian penduline tits may not be able to assess the risk of cuckoldry; thus, a direct association with parental care is unlikely to evolve. However, timing of desertion (i.e., when to desert during the egg-laying phase) may be influenced by the risk of cuckoldry. Future work applying extensive gene sequencing and quantitative genetics is likely to further our understanding of how selection may influence the association between parentage and parental care.

Glu415 in the alpha-tubulins plays a key role in stabilizing the microtubule-ADP-kinesin complexes.

Kavar(21g), a dominant female-sterile mutation of Drosophila, identifies the alphaTubulin67C gene that encodes alpha4-tubulin, the maternally provided alpha-tubulin isoform. Although alpha4-tubulin is synthesized during oogenesis, its function is required only in the early cleavage embryos. However, once present in the developing oocyte, much of the alpha4-tubulin and the Kavar(21g)-encoded E426K-alpha4-tubulin molecules become incorporated into the microtubules. We analyzed ooplasmic streaming and lipid-droplet transport, with confocal reflection microscopy, in the developing egg primordia in the presence and absence of alpha4-tubulin and E426K-alpha4-tubulin and learnt that the E426K-alpha4-tubulin molecules eliminate ooplasmic streaming and alter lipid-droplet transport. Apparently, Glu426 is involved in stabilization of the microtubule-kinesin complexes when the kinesins are in the most labile, ADP-bound state. Replacement of Glu426 by Lys results in frequent detachments of the kinesins from the microtubules leading to reduced transport efficiency and death of the embryos derived from the Kavar(21g)-carrying females. Glu426 is a component of the twelfth alpha-helix, which is the landing and binding platform for the mechanoenzymes. Since the twelfth alpha-helix is highly conserved in the alpha-tubulin family, Glu415, which corresponds to Glu426 in the constitutively expressed alpha-tubulins, seems be a key component of microtubule-kinesin interaction and thus the microtubule-based transport.

Nuclear localization signal-dependent and -independent movements of Drosophila melanogaster dUTPase isoforms during nuclear cleavage.

Two dUTPase isoforms (23 kDa and 21 kDa) are present in the fruitfly with the sole difference of an N-terminal extension. In Drosophila embryo, both isoforms are detected inside the nucleus. Here, we investigated the function of the N-terminal segment using eYFP-dUTPase constructs. In Schneider 2 cells, only the 23 kDa construct showed nuclear localization arguing that it may contain a nuclear localization signal (NLS). Sequence comparisons identified a lysine-rich nonapeptide with similarity to the human c-myc NLS. In Drosophila embryos during nuclear cleavages, the 23 kDa isoform showed the expected localization shifts. Contrariwise, although the 21 kDa isoform was excluded from the nuclei during interphase, it was shifted to the nucleus during prophase and forthcoming mitotic steps. The observed dynamic localization character showed strict timing to the nuclear cleavage phases and explained how both isoforms can be present within the nuclear microenvironment, although at different stages of cell cycle.

In vivo analysis of MT-based vesicle transport by confocal reflection microscopy.

The use of confocal reflection microscopy (CRM) for the in vivo analysis of microtubule (MT) mediated transport of lipid droplets in the developing Drosophila egg primordia is described here. The developing Drosophila oocytes are ideal objects to study MT-mediated transport in vivo: transport of e.g. the lipid droplets can be conveniently, selectively and sensitively monitored through CRM and the egg primordia are readily available for physical, chemical and/or genetic manipulations. CRM is a non-destructive way to follow vesicle movement and allows high frame rate image recording. When combined with fluorescence imaging, CRM offers simultaneous visualization of the cargo and the protein(s) of interest, i.e. a motor or a cargo adapter, thus allowing a better understanding of MT-mediated transport and spatiotemporal coordination of the transport machinery.

HorkaD, a chromosome instability-causing mutation in Drosophila, is a dominant-negative allele of Lodestar.

Correct segregation of chromosomes is particularly challenging during the rapid nuclear divisions of early embryogenesis. This process is disrupted by Horka(D), a dominant-negative mutation in Drosophila melanogaster that causes female sterility due to chromosome tangling and nondisjunction during oogenesis and early embryogenesis. Horka(D) also renders chromosomes unstable during spermatogenesis, which leads to the formation of diplo//haplo mosaics, including the gynandromorphs. Complete loss of gene function brings about maternal-effect lethality: embryos of the females without the Horka(D)-identified gene perish due to disrupted centrosome function, defective spindle assembly, formation of chromatin bridges, and abnormal chromosome segregation during the cleavage divisions. These defects are indicators of mitotic catastrophe and suggest that the gene product acts during the meiotic and the cleavage divisions, an idea that is supported by the observation that germ-line chimeras exhibit excessive germ-line and cleavage function. The gene affected by the Horka(D) mutation is lodestar, a member of the helicase-related genes. The Horka(D) mutation results in replacement of Ala777 with Thr, which we suggest causes chromosome instability by increasing the affinity of Lodestar for chromatin.

The DRE motif is a key component in the expression regulation of the importin-beta encoding Ketel gene in Drosophila.

Importin-beta, encoded by the Ketel gene in Drosophila, is a key component of nuclear protein import, the formation of the spindle microtubules and the assembly of the nuclear envelope. The Drosophila embryos rely on the maternal importin-beta dowry at the beginning of their life. Expression of the zygotic Ketel gene commences during gastrulation in every cell and while the expression is maintained in the mitotically active diploid cells it ceases in the non-dividing larval cells in which nuclear protein import is assured by the long persisting importin-beta molecules. How is the expression of the Ketel gene regulated? In silico analysis revealed several conserved transcription factor binding sequences in the Ketel gene promoter. Reporter genes in which different segments of the promoter ensured transient expression of the luciferase gene in S2 cells identified the sequences required for normal Ketel gene expression level. Gel retardation and band shift assays revealed that the DREF and the CFDD transcription factors play key roles in the regulation of Ketel gene expression. Transgenic LacZ reporter genes revealed the sequences that ensure tissue-specific gene expression. Apparently, the regulation of Ketel gene expression depends largely on a DRE motif and action of the DREF, CFDD, CF2-II and BEAF transcription factors.

Long persistence of importin-beta explains extended survival of cells and zygotes that lack the encoding gene.

Importin-beta is an essential component of nuclear protein import, spindle formation and nuclear envelope assembly. Formerly, the function of the Drosophila Ketel gene, which encodes importin-beta and is essential for the survival to adulthood, seemed to be required only in the mitotically active cells. We report here that importin-beta function is required in every cell and that this protein possesses an exceptionally long life span. Mosaic analysis, using gynanders, indicated that zygotic function of the Ketel gene is essential in a large group of cells in the embryos. Expression of a UAS-Ketel transgene by different tissue specific Gal4 drivers on ketel(null)/- hemizygous background revealed the requirement of Ketel gene function in the ectoderm. Elimination of the Ketel gene function using a UAS-Ketel-RNAi transgene driven by different Gal4 drivers confirmed the indispensability of the Ketel gene in the ectoderm. Using GFP-tagged importin-beta (encoded by a ketel(GFP) allele) we revealed that the maternally provided GFP-importin-beta molecules persist up to larval life. The zygotic Ketel gene is expressed in every cell during early gastrulation. Although the gene is then turned off in the non-dividing cells, the produced importin-beta molecules persist long and carry out nuclear protein import throughout the subsequent stages of development. In the continuously dividing diploid cells, the Ketel gene is constitutively expressed to fulfill all three functions of importin-beta.

alpha4-Tubulin is involved in rapid formation of long microtubules to push apart the daughter centrosomes during earlyx Drosophila embryogenesis.

Although alpha4-tubulin comprises only about one-fifth of the alpha-tubulin pool in every Drosophila egg, in the absence of alpha4-tubulin - in eggs of the kavar(0)/- hemizygous females - only a tassel of short microtubules forms with two barely separated daughter centrosomes. We report that alpha4-tubulin is enriched in the long microtubules that embrace the nuclear envelope and suggest that they push apart daughter centrosomes along the nuclear perimeter during the initial cleavage divisions. In vitro tubulin polymerization showed that alpha4-tubulin is required for rapid tubulin polymerization. Since tubulin polymerization is slow inside eggs of the kavar(0)/- females, only short microtubules can form within the 4 to 5 minutes allowed for the process. A tassel of short microtubules with two barely separated centrosomes forms in every egg of the Kavar(18c)/+ females, in which the cytoplasm contains both wild-type and Kavar(18c)-encoded alpha4-tubulin with an E82K amino acid substitution (E82K-alpha4-tubulin). E82K-alpha4-tubulin is incorporated into the microtubules and renders them unstable. When injected into wild-type early cleavage embryos E82K-alpha4-tubulin slows down the formation of long microtubules and the separation of the daughter centrosomes. Surprisingly, when injected into late cleavage embryos E82K-alpha4-tubulin is non-toxic. Similarly, in the neuroblasts, ectopically expressed E82K-alpha4-tubulin becomes incorporated into the microtubules that grow sufficiently long and function normally.

Gene expression profiling of human cardiac potassium and sodium channels.

BACKGROUND: The native cardiac ion currents and the action potential itself are the results of the concerted action of several different ion channels. The electrophysiological properties of cardiac cells are determined by the composition of ion channels and by their absolute abundance and proportional ratio. METHODS: Our aim in this study was to compare the gene expression level of a representative panel of cardiac ion channels with each other and to compare the same channels in the atrium and ventricle of the human heart using quantitative real-time PCR analysis. RESULTS: We obtained a significant difference in the gene expression levels in 21 of 35 channels between atrium and ventricle of healthy human hearts. Further, we found that the expression levels of Kv1.5 and Kv2.1 transcripts in the ventricle were very high, and that mRNAs for Kv1.7 and Kv3.4 are highly abundant in both the atrium and ventricle, which might indicate a functional role of these ion channel subunits in the formation of action potential in the human ventricle and both in the atrium and ventricle, respectively. CONCLUSIONS: This is the first report on the expression of several ion channel subunits, such as Kv1.7, Kv3.3 or Kv3.4 in human cardiomyocytes. The expression levels of these genes are comparable with that of well known ion channel subunits. Therefore, it is reasonable to assume, that these ion channel subunits may contribute to native currents in the human myocardium.

The homologous Drosophila transcriptional adaptors ADA2a and ADA2b are both required for normal development but have different functions.

In Drosophila and several other metazoan organisms, there are two genes that encode related but distinct homologs of ADA2-type transcriptional adaptors. Here we describe mutations of the two Ada2 genes of Drosophila melanogaster. By using mutant Drosophila lines, which allow the functional study of individual ADA2s, we demonstrate that both Drosophila Ada2 genes are essential. Ada2a and Ada2b null homozygotes are late-larva and late-pupa lethal, respectively. Double mutants have a phenotype identical to that of the Ada2a mutant. The overproduction of ADA2a protein from transgenes cannot rescue the defects resulting from the loss of Ada2b, nor does complementation work vice versa, indicating that the two Ada2 genes of Drosophila have different functions. An analysis of germ line mosaics generated by pole-cell transplantation revealed that the Ada2a function (similar to that reported for Ada2b) is required in the female germ line. A loss of the function of either of the Ada2 genes interferes with cell proliferation. Interestingly, the Ada2b null mutation reduces histone H3 K14 and H3 K9 acetylation and changes TAF10 localization, while the Ada2a null mutation does not. Moreover, the two ADA2s are differently required for the expression of the rosy gene, involved in eye pigment production, and for Dmp53-mediated apoptosis. The data presented here demonstrate that the two genes encoding homologous transcriptional adaptor ADA2 proteins in Drosophila are both essential but are functionally distinct.

The Kavar(D) dominant female-sterile mutations of Drosophila reveal a role for the maternally provided alpha-tubulin4 isoform in cleavage spindle maintenance and elongation.

The dominant-negative female-sterile Kavar(D) mutations and their revertant kavar(r) alleles identify the alphaTubulin67C gene of Drosophila melanogaster, which codes for the maternally provided alpha-tubulin(4) isoform. The mutations result in the formation of monopolar, collapsed spindles (each with two nearby centrosomes, a tassel of microtubules and overcondensed chromosomes), thus revealing a novel function for alpha-tubulin(4) in spindle maintenance and elongation. Molecular features of the two Kavar(D) alleles and a kavar(null) allele are described and models for their actions are discussed.