We describe the molecular characterization of the Drosophila melanogaster gene stubarista (sta) that encodes the highly conserved putative ribosome-associated protein D-p40. sta maps to cytological position 2A3-B2 on the X chromosome and encodes a protein (D-p40) of 270 amino acids. D-p40 shares 63% identity with the human p40 ribosomal protein. P element-mediated transformation of a 4.4-kb genomic fragment encompassing the 1-kb transcript corresponding to D-p40 was used to rescue both a lethal (sta2) and a viable (sta1) mutation at the stubarista (sta) locus. Developmental analysis of the sta2 mutation implicates a requirement for D-p40 during oogenesis and imaginal development, which is consistent with the expression of sta throughout development. In addition, we have analyzed the basis of the sta1 visible phenotype which consists of shortened antennae and bristles. sta1 is a translocation of the 1E1-2 to 2B3-4 region of the X chromosome onto the third chromosome at 89B21-C4. We provide genetic evidence that Dp(1;3)sta1 is mutant at the spineless (ss) locus and that it is associated with partial D-p40 activity. We demonstrate that sta1 acts as a recessive enhancer of ss; reduction in the amount of D-p40 provided by the transposed X chromosomal region of sta1 reveals a haplo-insufficient phenotype of the otherwise recessive ss mutations. This phenomenon is reminiscent of the enhancing effect observed with Minute mutations, one of which, rp49, has previously been shown to encode a ribosomal protein.
We have characterized the molecular nature of mutations in wingless (wg), a segment polarity gene acting during various stages of Drosophila development. Embryo-lethal alleles have undergone mutations in the protein-encoding domain of the gene, including deletions and point mutations of conserved residues. In a temperature sensitive mutation, a conserved cysteine residue is replaced by a serine. In embryo-viable alleles, the wg transcriptional unit is not affected. Immunostaining of mutant embryos shows that the embryo-lethal alleles produce either no wg antigen or a form of the protein that is retained within cells. Interestingly, embryos mutant for the segment polarity gene porcupine show a similar retention of the wg antigen. We have also transfected wild type wg alleles into Drosophila tissue culture cells, which then display wg protein on the cell surface and in the extracellular matrix. In similar experiments with mutant alleles, the proteins are retained in intracellular compartments and appear not to be secreted. These data provide further evidence that wg acts as a secreted factor and suggest that porcupine provides an accessory function for wg protein secretion or transport.
BACKGROUND: Cell lineage analysis and mosaic analysis of mutations are important techniques that are used to study the development of many organisms. Unfortunately, the methods employed for such analyses are usually inefficient, technically demanding or labor intensive. In Drosophila, the most common methodology used for the generation of mosaic animals is mitotic recombination, which is induced by X-rays. Although this technique is simple, it has the undesirable characteristics of a low efficiency and a high rate of cell death. Furthermore, although a large number of marker systems has been employed to detect mitotic recombinants, none allows easy identification of clones for all cell types. RESULTS: A system is described here that allows a highly efficient generation of clones with the concomitant expression of an easily detectable cellular marker. This method can be applied to cell lineage and mosaic analysis in Drosophila. The site-specific yeast FLP recombinase, under the control of a heat shock-inducible promoter, efficiently catalyses mitotic recombination specifically at the site of a FLP recombination target (FRT). In this system, recombination fuses the alpha-tubulin promoter to the lacZ gene, allowing transcription of the marker. Recombinant cells and their progeny can, therefore, be detected by standard assays for beta-galactosidase. Of particular importance is the fact that only the cells of interest stain, thus allowing their simple detection in any tissue. CONCLUSIONS: We demonstrate that, by intermolecular recombination, we can use FLIP recombinase to generate marked clones efficiently in embryonic, larval and adult tissues. This simple and efficient technique is well suited to cell-lineage analysis and can be easily extended to the generation and detection of mutant clones in mosaic animals.
We have designed a system for targeted gene expression that allows the selective activation of any cloned gene in a wide variety of tissue- and cell-specific patterns. The gene encoding the yeast transcriptional activator GAL4 is inserted randomly into the Drosophila genome to drive GAL4 expression from one of a diverse array of genomic enhancers. It is then possible to introduce a gene containing GAL4 binding sites within its promoter, to activate it in those cells where GAL4 is expressed, and to observe the effect of this directed misexpression on development. We have used GAL4-directed transcription to expand the domain of embryonic expression of the homeobox protein even-skipped. We show that even-skipped represses wingless and transforms cells that would normally secrete naked cuticle into denticle secreting cells. The GAL4 system can thus be used to study regulatory interactions during embryonic development. In adults, targeted expression can be used to generate dominant phenotypes for use in genetic screens. We have directed expression of an activated form of the Dras2 protein, resulting in dominant eye and wing defects that can be used in screens to identify other members of the Dras2 signal transduction pathway.
Many of the steps involved in formation of the Drosophila embryonic central nervous system (CNS) have been identified by both descriptive and experimental studies. In this review we will describe the various approaches that have been used to identify molecules involved in CNS development and the advantages and disadvantages of each of them. Our discussion will by no means be exhaustive; but rather we will discuss our experiences with each approach and provide an overview of what has been learned by using these methodologies. Finally, we will discuss methods that have been recently developed and how they are likely to provide further insight into CNS development.
By a complex and little understood mechanism, segment polarity genes control patterning in each segment of the Drosophila embryo. During this process, cell to cell communication plays a pivotal role and is under direct control of the products of segment polarity genes. Many of the cloned segment polarity genes have been found to be highly conserved in evolution, providing a model system for cellular interactions in other organisms. In Drosophila, two of these genes, engrailed and wingless, are expressed on either side of the parasegment border. wingless encodes a secreted molecule and engrailed a nuclear protein with a homeobox. Maintenance of engrailed expression is dependent on wingless and vice versa. To investigate the role of other segment polarity genes in the mutual control between these two genes, we have examined wingless and engrailed protein distribution in embryos mutant for each of the segment polarity genes. In embryos mutant for armadillo, dishevelled and porcupine, the changes in engrailed expression are identical to those in wingless mutant embryos, suggesting that their gene products act in the wingless pathway. In embryos mutant for hedgehog, fused, cubitus interruptus Dominant and gooseberry, expression of engrailed is affected to varying degrees. However wingless expression in the latter group decays in a similar way earlier than engrailed expression, indicating that these gene products might function in the maintenance of wingless expression. Using double mutant embryos, epistatic relationships between some segment polarity genes have been established. We present a model showing a current view of segment polarity gene interactions.
In the Drosophila embryo, specification of terminal cell fates that result in the formation of both the head (acron) and tail (telson) regions is under the control of the torso (tor) receptor tyrosine kinase. The current knowledge suggests that activation of tor at the egg pole initiates a signal transduction pathway that is mediated sequentially by the guanine nucleotide releasing factor son of sevenless (Sos), the p21Ras1 GTPase, the serine/threonine kinase D-raf and the tyrosine/threonine kinase MAPKK (Dsor1). Subsequently, it is postulated that activation, possibly by phosphorylation, of a transcription factor at the egg poles activates the transcription of the terminal gap genes tailless and huckebein. These gap genes, which encode putative transcription factors, then control the expression of more downstream factors that ultimately result in head and tail differentiation. Also involved in tor signaling is the non-receptor protein tyrosine phosphatase corkscrew (csw). Here, we review the current model and discuss future research directions in this field.
We describe the characterization of the Drosophila gene, corkscrew (csw), which is maternally required for normal determination of cell fates at the termini of the embryo. Determination of terminal cell fates is mediated by a signal transduction pathway that involves a receptor tyrosine kinase, torso, a serine/threonine kinase, D-raf, and the transcription factors, tailless and huckebein. Double mutant and cellular analyses between csw, torso, D-raf, and tailless indicate that csw acts downstream of torso and in concert with D-raf to positively transduce the torso signal via tailless, to downstream terminal genes. The csw gene encodes a putative nonreceptor protein tyrosine phosphatase covalently linked to two N-terminal SH2 domains, which is similar to the mammalian PTP1C protein.
We describe the molecular characterization of the Drosophila gene spitz (spi), which encodes a putative 26-kD, EGF-like transmembrane protein that is structurally similar to TGF-alpha. Temporal and spatial expression patterns of spi transcripts indicate that spi is expressed throughout the embryo. Examination of mutant embryos reveals that spi is involved in a number of unrelated developmental choices, for example, dorsal-ventral axis formation, glial migration, sensory organ determination, and muscle development. We propose that spi may act as a ligand for cell-specific receptors, possibly rhomboid and/or the Drosophila EGF receptor homolog.
Lethal alleles of orthodenticle (= otd) cause abnormalities in the embryonic head that reflect an early role in anterior pattern formation. In addition, otd activity is required for the development of the larval and adult epidermis. Clonal analysis of both viable and lethal alleles shows that the adult requirement for otd is restricted to medial regions of certain discs. When otd activity is reduced or removed, some medial precursor cells produce bristles and cuticle characteristic of more lateral structures. Similar medial defects are observed in the larval epidermis of embryos homozygous for lethal otd alleles. Antibodies to otd recognize a nuclear protein found at high levels in the medial region of the eye antennal discs, the leg discs, the genital discs and along the ventral midline of the ventral epidermis of the embryo. These results suggest that the otd gene product is required to specify medial cell fates in both the larval and adult epidermis.
An efficient method for generating embryonic mosaics using a yeast site-specific recombinase (FLP), under the control of a heat shock promoter, is described. FLP-recombinase can promote mitotic exchange between homologous chromosomes that contain FRT (FLP Recombination Target) sequences. To demonstrate the efficiency of FLP-recombinase to generate embryonic mosaics, clones of the recessive and cell autonomous mutation armadillo (arm), detected by their ability to differentiate ectopic denticles in the naked cuticle of each abdominal segment, have been induced. We have analyzed the parameters of FLP-recombinase induced embryonic mitotic recombination and have demonstrated that clones can be efficiently induced during the postblastoderm mitotic divisions. We discuss applications of this technique for the analyses of the roles of various mutations during embryonic patterning.
Intrasegmental patterning in the Drosophila embryo is regulated by cell-cell communication. One of the signaling pathways that operates to specify positional information throughout the segment is mediated by the wingless (wg) protein, which is the homolog of the proto-oncogene Wnt-1. The early role of wg is to stabilize engrailed (en) expression by initiating a phase of en autoregulation in the adjacent more posterior cells. Here, we report that the segment polarity gene zeste-white 3 (zw3; also known as shaggy) acts as a repressor of en autoregulation. Genetic epistasis experiments indicate that wg signaling operates by inactivating the zw3 repression of en autoactivation. In addition, we demonstrate that zw3 encodes the Drosophila homolog of mammalian glycogen synthase kinase-3.
Vectors derived from the Drosophila P element transposon are widely used to make transgenic Drosophila. Insertion of most P-element-derived vectors is nonrandom, but they exhibit a broad specificity of target sites. During experiments to identify cis-acting regulatory elements of the Drosophila segmentation gene engrailed, we identified a fragment of engrailed DNA that, when included within a P-element vector, strikingly alters the specificity of target sites. P-element vectors that contain this fragment of engrailed regulatory DNA insert at a high frequency near genes expressed in stripes.
Polytene section 17 of the X chromosome of Drosophila melanogaster, previously known to contain six putative lethal complementation groups important in oogenesis and embryogenesis, has here been further characterized genetically and developmentally. We constructed fcl+Y, a duplication of this region, which allowed us to conduct mutagenesis screens specific for the region and to perform complementation analyses (previously not possible). We recovered 67 new lethal mutations which defined 15 complementation groups within Df(1)N19 which deletes most of polytene section 17. The zygotic lethal phenotypes of these and preexisting mutations within polytene section 17 were examined, and their maternal requirements were analysed in homozygous germline clones using the dominant female sterile technique. We present evidence that an additional gene, which produces two developmentally regulated transcripts, is located in this region and is involved in embryogenesis, although no mutations in this gene were identified. In this interval of 37 to 43 polytene chromosome bands we have defined 17 genes, 12 (71%) of which are of significance to oogenesis or embryogenesis.
We describe an efficient method for generating female germline mosaics by inducing site-specific homologous mitotic recombination with a yeast recombinase (FLP) which is driven by a heat shock promoter. These germline mosaics are produced in flies heterozygous for the agametic, germline-dependent, dominant female sterile (DFS) mutation ovoD1, where only flies possessing germline clones are able to lay eggs. This method, the "FLP-DFS" technique, is very efficient because more than 90% of females with germline clones can be recovered. We show that this heat-inducible, site-specific mitotic recombination system does not affect viability and that the germline clones recovered are physiologically the same as those created by X-ray induced mitotic recombination. We describe the parameters of FLP-recombinase induced germline mitotic recombination and the use of the "FLP-DFS" technique to analyze the maternal effect of X-linked zygotic lethal mutations.
The formation of the telson in the Drosophila embryo, which encompasses all structures posterior to abdominal segment 7, is under the control of the "terminal class" genes. These maternally expressed genes are organized in a signal transduction pathway which implicates cell-cell interactions between the germ cell derivatives (the nurse cells and oocyte) and the surrounding follicle cell epithelium. Activation of this localized signal transduction pathway at the termini of the embryo is believed to specify the domains of activation and repression of a set of zygotic genes whose interactions specify the various cell states required for the proper formation of tail structures.
The crooked neck (crn) gene of Drosophila encodes a protein of 702 amino acids and contains 16 tandemly arranged copies of a 34-amino-acid repeat that is similar to the tetratrico peptide repeat (TPR). Multiple copies of the TPR motif have also been found in a family of yeast genes, including several members that are necessary for cell division. TPR-containing proteins encoded by the yeast genes CDC16, CDC23, and nuc2+ are required for progression through the G2/M transition of the cell cycle. Loss of zygotic expression of crn causes defects in the proliferation of brain neuroblasts and results in the absence of identified neuronal lineages in the central and peripheral nervous systems. The sequence similarity and mutant phenotypes are consistent with a cell cycle requirement for the crn gene product.
To generate cell- and tissue-specific expression patterns of the reporter gene lacZ in Drosophila, we have generated and characterized 1,426 independent insertion strains using four different P-element constructs. These four transposons carry a lacZ gene driven either by the weak promoter of the P-element transposase gene or by partial promoters from the even-skipped, fushi-tarazu, or engrailed genes. The tissue-specific patterns of beta-galactosidase expression that we are able to generate depend on the promoter utilized. We describe in detail 13 strains that can be used to follow specific cell lineages and demonstrate their utility in analyzing the phenotypes of developmental mutants. Insertion strains generated with P-elements that carry various sequences upstream of the lacZ gene exhibit an increased variety of expression patterns that can be used to study Drosophila development.