A selective screen reveals discrete functional domains in Drosophila Nanos

The Drosophila protein Nanos encodes an evolutionarily conserved protein with two zinc finger motifs. In the embryo, Nanos protein function is required for establishment of the anterior-posterior body pattern and for the migration of primordial germ cells. During oogenesis, Nanos protein is involved in the establishment and maintenance of germ-line stem cells and the differentiation of oocyte precursor cells. To establish proper embryonic patterning, Nanos acts as a translational regulator of hunchback RNA. Nanos' targets for germ cell migration and development are not known. Here, we describe a selective genetic screen aimed at isolating new nanos alleles. The molecular and genetic analysis of 68 new alleles has allowed us to identify amino acids critical for nanos function. This analysis shows that the CCHC motifs, which coordinate two metal ions, are essential for all known functions of Nanos protein. Furthermore, a region C-terminal to the zinc fingers seems to constitute a novel functional domain within the Nanos protein. This "tail region" of Nanos is required for abdomen formation and germ cell migration, but not for oogenesis.     

Genetic analysis of new mutations affecting the phenotypic manifestations of alleles from the achaete-scute complex in Drosophila melanogaster

The achaete-scute complex contains four homologous genes involved in formation of the central and periphery nervous system in Drosophila melanogaster. The achaete and scute mutations lead to reduction of bristles and hairs on head and thorax. We found mutations at three loci not analyzed earlier. The suppressor of scute mutations suppress phenotypic expression of the achaete and scute alleles. Mutations in two other genes, pseudoscute and microchaetae, induce bristles reduction, i.e. the changes typical of scute mutations. Possible role of these genes in development of nervous system is being discussed.     

A spatial map of olfactory receptor expression in the Drosophila antenna

Insects provide an attractive system for the study of olfactory sensory perception. We have identified a novel family of seven transmembrane domain proteins, encoded by 100 to 200 genes, that is likely to represent the family of Drosophila odorant receptors. Members of this gene family are expressed in topographically defined subpopulations of olfactory sensory neurons in either the antenna or the maxillary palp. Sensory neurons express different complements of receptor genes, such that individual neurons are functionally distinct. The isolation of candidate odorant receptor genes along with a genetic analysis of olfactory-driven behavior in insects may ultimately afford a system to understand the mechanistic link between odor recognition and behavior.     

Patterning of the Drosophila nervous system: the achaete-scute gene complex.

The genes of the achaete-scute complex (AS-C) confer on cells the ability to become neural precursors. Their expression is restricted to groups of cells, the proneural clusters, which occupy specific positions within the embryo neural anlagen and the larva imaginal discs. Neuroblasts or sensory organ mother cells are born within these clusters. Thus, the patterns of expression of the AS-C genes help to define the topology of the nervous system.     

Genetic analysis of viable Hsp90 alleles reveals a critical role in Drosophila spermatogenesis

The Hsp90 chaperone protein maintains the activities of a remarkable variety of signal transducers, but its most critical functions in the context of the whole organism are unknown. Point mutations of Hsp83 (the Drosophila Hsp90 gene) obtained in two different screens are lethal as homozygotes. We report that eight transheterozygous mutant combinations produce viable adults. All exhibit the same developmental defects: sterile males and sterile or weakly fertile females. We also report that scratch, a previously identified male-sterile mutation, is an allele of Hsp82 with a P-element insertion in the intron that reduces expression. Thus, it is a simple reduction in Hsp90 function, rather than possible altered functions in the point mutants, that leads to male sterility. As shown by light and electron microscopy, all stages of spermatogenesis involving microtubule function are affected, from early mitotic divisions to later stages of sperm maturation, individualization, and motility. Aberrant microtubules are prominent in yeast cells carrying mutations in HSP82 (the yeast Hsp90 gene), confirming that Hsp90 function is connected to microtubule dynamics and that this connection is highly conserved. A small fraction of Hsp90 copurifies with taxol-stabilized microtubule proteins in Drosophila embryo extracts, but Hsp90 does not remain associated with microtubules through repeated temperature-induced assembly and disassembly reactions. If the spermatogenesis phenotypes are due to defects in microtubule dynamics, we suggest these are indirect, reflecting a role for Hsp90 in maintaining critical signal transduction pathways and microtubule effectors, rather than a direct role in the assembly and disassembly of microtubules themselves.     

Adaptation of Drosophila to a novel laboratory environment reveals temporally heterogeneous trajectories of selected alleles

The genomic basis of adaptation to novel environments is a fundamental problem in evolutionary biology that has gained additional importance in the light of the recent global change discussion. Here, we combined laboratory natural selection (experimental evolution) in Drosophila melanogaster with genome‐wide next generation sequencing of DNA pools (Pool‐Seq) to identify alleles that are favourable in a novel laboratory environment and traced their trajectories during the adaptive process. Already after 15 generations, we identified a pronounced genomic response to selection, with almost 5000 single nucleotide polymorphisms (SNP; genome‐wide false discovery rates < 0.005%) deviating from neutral expectation. Importantly, the evolutionary trajectories of the selected alleles were heterogeneous, with the alleles falling into two distinct classes: (i) alleles that continuously rise in frequency; and (ii) alleles that at first increase rapidly but whose frequencies then reach a plateau. Our data thus suggest that the genomic response to selection can involve a large number of selected SNPs that show unexpectedly complex evolutionary trajectories, possibly due to nonadditive effects.     

A role for the segment polarity gene shaggy/GSK-3 in the Drosophila circadian clock.

Tissue-specific overexpression of the glycogen synthase kinase-3 (GSK-3) ortholog shaggy (sgg) shortens the period of the Drosophila circadian locomotor activity cycle. The short period phenotype was attributed to premature nuclear translocation of the PERIOD/TIMELESS heterodimer. Reducing SGG/GSK-3 activity lengthens period, demonstrating an intrinsic role for the kinase in circadian rhythmicity. Lowered sgg activity decreased TIMELESS phosphorylation, and it was found that GSK-3 beta specifically phosphorylates TIMELESS in vitro. Overexpression of sgg in vivo converts hypophosphorylated TIMELESS to a hyperphosphorylated protein whose electrophoretic mobility, and light and phosphatase sensitivity, are indistinguishable from the rhythmically produced hyperphosphorylated TIMELESS of wild-type flies. Our results indicate a role for SGG/GSK-3 in TIMELESS phosphorylation and in the regulated nuclear translocation of the PERIOD/TIMELESS heterodimer.     

A BAC transgenic Hes1-EGFP reporter reveals novel expression domains in mouse embryos

Expression of the basic helix-loop-helix factor Hairy and Enhancer of Split-1 (Hes1) is required for normal development of a number of tissues during embryonic development. Depending on context, Hes1 may act as a Notch signalling effector which promotes the undifferentiated and proliferative state of progenitor cells, but increasing evidence also points to Notch independent regulation of Hes1 expression. Here we use high resolution confocal scanning of EGFP in a novel BAC transgenic mouse reporter line, Tg(Hes1-EGFP)^1Hri, to analyse Hes1 expression from embryonic day 7.0 (e7.0). Our data recapitulates some previous observations on Hes1 expression and suggests new, hitherto unrecognised expression domains including expression in the definitive endoderm at early somite stages before gut tube closure and thus preceding organogenesis. This mouse line will be a valuable tool for studies addressing the role of Hes1 in a number of different research areas including organ specification, development and regeneration.     

Rapid divergence in the course of Drosophila evolution reveals structural important domains of the Notch antagonist Hairless

 Hairless is a member of the Notch signalling pathway, where it acts as antagonist by binding to Suppressor of Hairless [Su(H)], thereby inhibiting Notch target gene activation. The pathway and its members are highly conserved in metazoans from worms to humans. However, a Hairless orthologue from another species has not yet been identified. The identification of Hairless in largely diverged species by cross-hybridization has failed so far probably due to a low degree of conservation. Therefore, we turned to D. hydei where a Hairless mutation has been described before. The D. hydei Hairless orthologue is reasonably well conserved with regard to gene structure and expression. The prospective Hairless protein orthologues share several highly conserved regions which are separated by quite diverged stretches. As to be expected, the largest region of high conservation corresponds to the Su(H) binding domain. This region is also functionally conserved, since this D. hydei protein domain binds very strongly to the D. melanogaster Su(H) protein. The other conserved regions support our earlier structure-function analysis since they nicely correspond to previously defined, functionally important protein domains. Most notably, the very C-terminal domain which is very sensitive to structural alterations, is nearly identical between the two species. In summary, this evolutionary study improves the knowledge on functionally significant domains of the Hairless protein, and may be helpful for the future identification of homologues in other animals, especially in vertebrates. Received: 26 August 1998 / Accepted: 9 November 1998     

Transcriptional activation by heterodimers of the achaete-scute and daughterless gene products of Drosophila

The achaete-scute complex (AS-C) and the daughterless (da) genes encode helix-loop-helix proteins which have been shown to interact in vivo and to be required for neurogenesis. We show in vitro that heterodimers of three AS-C products with DA bind DNA strongly, whereas DA homodimers bind weakly and homo or heterocombinations of AS-C products not at all. Proteins unable to dimerize did not bind DNA. Target sequences for the heterodimers were found in the promoters of the hunchback and the achaete genes. Using sequences of the former we show that the DNA binding results obtained in vitro fully correlate with the ability of different combinations to activate the expression of a reporter gene in yeast. Embryos deficient for the lethal of scute gene fail to activate hunchback in some neural lineages in a pattern consistent with the lack of a member of a multigene family.     

A mutational analysis of dishevelled in Drosophila defines novel domains in the dishevelled protein as well as novel suppressing alleles of axin

Drosophila dishevelled (dsh) functions in two pathways: it is necessary to transduce Wingless (Wg) signaling and it is required in planar cell polarity. To learn more about how Dsh can discriminate between these functions, we performed genetic screens to isolate additional dsh alleles and we examined the potential role of protein phosphorylation by site-directed mutagenesis. We identified two alleles with point mutations in the Dsh DEP domain that specifically disrupt planar polarity signaling. When positioned in the structure of the DEP domain, these mutations are located close to each other and to a previously identified planar polarity mutation. In addition to the requirement for the DEP domain, we found that a cluster of potential phosphorylation sites in a binding domain for the protein kinase PAR-1 is also essential for planar polarity signaling. To identify regions of dsh that are necessary for Wg signaling, we screened for mutations that modified a GMR-GAL4;UAS-dsh overexpression phenotype in the eye. We recovered many alleles of the transgene containing missense mutations, including mutations in the DIX domain and in the DEP domain, the latter group mapping separately from the planar polarity mutations. In addition, several transgenes had mutations within a domain containing a consensus sequence for an SH3-binding protein. We also recovered second-site-suppressing mutations in axin, mapping at a region that may specifically interact with overexpressed Dsh.     

Developmental expression and spatial distribution of dopa decarboxylase in Drosophila

Regulation of the dopa decarboxylase gene of Drosophila has been studied at the genetic and molecular levels. Here we report a direct assay for the tissue and temporal regulation of Ddc. A dopa decarboxylase (DDC) peptide was obtained by bacterial expression of a portion of the DDC gene in a pUC plasmid. Antisera raised against this biologically purified DDC peptide react specifically with Drosophila DDC in histological preparations and protein blots. The levels of DDC cross-reacting material closely parallel the levels of enzyme activity observed during development, indicating that DDC is degraded during periods of declining activity. We find that DDC is expressed in only two tissues, namely, the epidermis and the nervous system of the larva and adult. Epidermal DDC was found within the epidermal cells and was not detected in the overlying cuticle. DDC-containing neurons were observed in the central as well as in the visceral nervous system. Paired and unpaired midline neurons in the ventral ganglia are arranged in a segmental pattern. A subset of the DDC-positive neurons appears to correlate with the serotonin-positive neurons suggesting that the others are producing only dopamine. We find that the DDC activity associated with the proventriculus and ovary is due to the presence of DDC in the stomatogastric and caudal system neurons specifically associated with those structures.     

A series of mutable alleles of the white locus in Drosophila melanogaster

Summary The origin and phenotypes of a number of zeste mutant stocks with mutable white loci are described. Each newly arising form was lighter in eye color than the mutant it originated from. In each case the lighter pigmentation is believed to be due to an increase in genetic material in the proximal region of the white locus, the increase supposedly being the result of unequal crossing over. Some of the mutations which arose in the mutable stocks are reversions. They occurred in males as well as in homo- and heterozygous females. The reversions are believed to be due to a decrease in genetic material in the proximal region of the white locus. The decrease is assumed to be the result of intrachromosomal recombination. At least some of these events took place premeiotically. New mutants which originate frequently from mutable stocks are stable. In addition to the structure of the mutable white locus there is probably at least one still unknown factor which affects its mutability since the frequency of mutations arising in the mutable stocks decreases over the years.     

A dosage-sensitive modifier of retrotransposon-induced alleles of the Drosophila white locus.

The apricot allele of the white locus results from the insertion of the retrotransposon copia. Mutations in a newly discovered locus, the Darkener-of-apricot (Doa), suppress wa and some of its revertants. Of 44 other white alleles tested, only wsp55 is affected by Doa, although, in contrast, it is enhanced by Doa mutations. The Doa locus modulates wa and wsp55 expression as a function of its own dosage. Mutations in Doa are dominant suppressors or enhancers and are recessive lethals. Rare Doa mutant homozygotes escaping lethality demonstrate extreme phenotypic suppression of wa and enhancement of wsp55. RNA from wa is substantially wild-type in structure in escapers, although reduced in quantity.     

Interacting insulators from the Drosophila melanogaster bithorax complex can form independent expression domains

Regulatory region of three bithorax complex genes, Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) can be divided into nine iab domains, capable of directing expression of one of the genes in certain abdominal parasegment of Drosophila. In the Abd-B regulatory region, three insulators were identified, including Fab-7 and Fab-8, which flanked the iab-7domain, and Mcp, which separated the Abd-B and abd-A regulatory regions. It was suggested that boundary insulators formed a barrier between active and repressed chromatin. In the present study, using the yellow and white reporter genes and different combinations of known insulators, Mcp, Fab-7, and Fab-8, it was demonstrated that only specific interaction of two insulators was capable of isolation of active and repressed chromatin, i.e., the formation of independent expression domains.