Analysis of a swallow homologue from Drosophila pseudoobscura

We analyzed a functional homologue of the swallow gene from Drosophila pseudoobscura. The swallow gene of D. melanogaster plays an essential role in localizing bicoid mRNA in oocytes, and swallow mutant embryos show anterior pattern defects that result from the lack of localization of the bicoid morphogen. The pseudoobscura homologue rescues the function of swallow mutants when introduced into the genome of D. melanogaster, and its expression is similar to that of the melanogaster gene. The predicted pseudoobscura and melanogaster proteins are 49% identical and 69% conserved. The coiled-coil domain previously identified in the melanogaster swallow protein is strongly conserved in the pseudoobscura homologue, but the weak similarity of the melanogaster swallow protein to the RNP class of RNA-binding proteins is not conserved in the pseudoobscura homologue. These and other observations suggest a structural role for swallow in localizing bicoid mRNA, perhaps as part of the egg cytoskeleton. Received: 3 August 1999 / Accepted: 29 September 1999     

Localization of swallow-Green Fluorescent Protein in Drosophila oogenesis and implications for the role of swallow in RNA localization

The localization of a hybrid protein composed of swallow and Green Fluorescent Protein (GFP) during Drosophila oogenesis is reported. I constructed a hybrid gene with GFP inserted into an internal position of swallow. This gene was integrated into the Drosophila genome and provides full swallow+ function, as assayed by the complete rescue of strong swallow mutants. Swallow-GFP is localized at all points along the oocyte cortex from vitellogenic stages of oogenesis through the end of oogenesis. Higher concentrations of swallow-GFP are present at the anterior oocyte cortex than at the lateral and posterior oocyte cortices at Stages 10 and 11, when bicoid and htsN4 mRNA transport from nurse cells and localization in the oocyte are most active. At Stage 9 and at Stages 12-14 swallow-GFP is equally distributed at the anterior, lateral, and posterior oocyte cortices. The position of swallow-GFP in vitellogenic stages is identical to the position of endogenous swallow protein determined by indirect immunofluorescence using an anti-swallow antibody. At the oocyte cortex, swallow-GFP is present in particulate structures that lie within or just internal to the dense cortical actin meshwork. These particles show little or no movement, suggesting that they are attached to or embedded in the oocyte cortex. These observations are most easily interpreted in the context of mRNA anchoring or microtubule organizing functions for the swallow protein.     

The yan gene is highly conserved in Drosophila and its expression suggests a complex role throughout development

 Competence for cell fate determination and cellular differentiation is under tight control of regulatory genes. Yan, a nuclear target of receptor tyrosine kinase (RTK) signaling, is an E twenty six (ETS) DNA-binding protein that functions as a negative regulator of cell differentiation and proliferation in Drosophila. Most members of RTK signaling pathways are highly conserved through evolution, yet no yan orthologues have been identified to date in vertebrates. To investigate the degree of yan conservation during evolution, we have characterized a yan homologue from a sibling species of D. melanogaster, D. virilis. Our results show that the organization, primary structure and expression pattern of yan are highly conserved. Both genes span over 20 kb and contain four exons with introns at identical positions. The areas with highest amino acid similarity include the Pointed and ETS domain but there are other discrete regions with a high degree of similarity. Phylogenetic analysis reveals that yan’s closest relative is the human tel gene, a negative regulator of differentiation in hematopoetic precursors. In both species, Yan is dynamically expressed beginning as early as stage 4/5 and persisting throughout embryogenesis. In third instar larvae, Yan is expressed in and behind the morphogenetic furrow of the eye imaginal disc as well as in the laminar precursor cells of the brain. Ovarian follicle cells also contain Yan protein. Conservation of the structure and expression patterns of yan genes strongly suggests that regulatory mechanisms for their expression are also conserved in these two species. Received: 3 November 1998 / Accepted: 9 December 1998     

Lineage and fate in Drosophila: role of the gene tramtrack in sense organ development

 The tactile bristles of the fly comprise four cells that originate from a single precursor cell through a fixed lineage. The gene tramtrack (ttk) plays a crucial role in defining the fates of these cells. Here we analyse the normal pattern of expression of ttk, as well as the effect of ttk overexpression at different steps of the lineage. We show that ttk is never expressed in cells having a neural potential, and that in cells where ttk is expressed, there is a delay between division and the onset of expression. The ectopic expression of ttk before some stage of the cell cycle can block further cell division. Furthermore, this expression transforms neural into non-neural cells, suggesting that ttk acts as a repressor of neural fate at each step of the lineage. Our results suggest that ttk is probably not involved in setting up the mechanism that creates an asymmetry between sister cells, but rather in the implementation of that choice. Received: 10 October 1996 / Accepted: 11 February 1997     

Dissection of cis-regulatory elements of the Drosophila gene Serrate

 The Drosophila gene Serrate encodes a membrane spanning protein, which is expressed in a complex pattern during embryogenesis and larval stages. Loss of Serrate function leads to larval lethality, which is associated with several morphogenetic defects, including the failure to develop wings and halteres. Serrate has been suggested to act as a short-range signal during wing development. It is required for the induction of the organising centre at the dorsal/ventral compartment boundary, from which growth and patterning of the wing is controlled. In order to understand the regulatory network required to control the spatially and temporally dynamic expression of Serrate, we analysed its cis-regulatory elements by fusing various genomic fragments upstream of the reporter gene lacZ. Enhancer elements reflecting the expression pattern of endogenous Serrate in embryonic and postembryonic tissues could be confined to 26 kb of genomic DNA, including 9 kb of transcribed region. Expression in some embryonic tissues is under the control of multiple enhancers located in the 5’ region and in intron sequences. The data presented here provide the tools to unravel the genetic network which regulates Serrate during different developmental stages in diverse tissues. Received: 27 March 1998 / Accepted: 17 May 1998     

The wingless gene is required for embryonic brain development in Drosophila

 We have studied the role of the wingless gene in embryonic brain development of Drosophila. wingless is expressed in a large domain in the anlage of the protocerebrum and also transiently in smaller domains in the anlagen of the deutocerebrum and tritocerebrum. Elimination of the wingless gene in null mutants has dramatic effects on the developing protocerebrum; although initially generated, approximately one half of the protocerebrum is deleted in wingless null mutants by apoptotic cell death at late embryonic stages. Using temperature sensitive mutants, a rescue of the mutant phenotype can be achieved by stage-specific expression of functional wingless protein during embryonic stages 9–10. This time period correlates with that of neuroblast specification but preceeds the generation and subsequent loss of protocerebral neurons. Ectopic wingless over-expression in gain-of-function mutants results in dramatically oversized CNS. We conclude that wingless is required for the development of the anterior protocerebral brain region in Drosophila. We propose that an important role of wingless in this part of the developing brain is the determination of neural cell fate. Received: 7 October 1997 / Accepted: 30 December 1997     

Molecular characterization of myoplasmin-C1: a cytoskeletal component localized in the myoplasm of the ascidian egg

 Myoplasmin-C1 is a polypeptide detected by a monoclonal antibody, which is localized in the myoplasm of ascidian eggs. Since microinjection of the antibody blocks larval muscle development, myoplasmin-C1 may play a role in muscle cell differentiation (Nishikata et al. 1987). Isolation and characterization of myoplasmin-C1 cDNA clones revealed that the predicted amino acid sequence of myoplasmin-C1 had no similarity to any known protein. However, the deduced protein contains heptad repeats similar to those in myosin heavy chain, tropomyosin and the Drosophila Bicaudal D gene product, suggesting that it is a filamentous component of the myoplasmic cytoskeleton. The predicted amino acid sequence also showed several possible phosphorylation sites. Consistent with the prediction that myoplasmin-C1 is a cytoskeletal component, the protein remained in the myoplasmic cytoskeletal domain after detergent extraction. These results suggest that myoplasmin-C1 is a cytoskeletal component of the myoplasm and that it plays a role in anchoring and segregating muscle determinants. Received: 6 October 1995 / Accepted in revised form: 7 December 1995     

Expression of the PI4P 5-kinase Drosophila homologue skittles in the germline suggests a role in spermatogenesis and oogenesis

 We have isolated the Drosophila gene skittles (sktl) which shows homology to members of a novel family of phosphatidylinositol-4-phosphate 5-kinases, including the gene product encoded by the human STM-7.I gene which has been assigned to the neurodegenerative disorder Friedreichs ataxia. In situ hybridization reveals sktl expression during oogenesis and spermatogenesis. Received: 7 February1997 / Accepted: 4 April 1997     

2D gene expression parameters of wing imaginal disc of Drosophila for developmental analysis

 By using high resolution two-dimensional (2D) gel electrophoresis coupled with computer-analysis we have established a quantitative Drosophila wing imaginal disc protein database of third instar larvae as a reference to be used for comparative purposes in genetic studies. A general catalogue integrated by 1,184 ³⁵S-methionine-labelled polypeptides from wing imaginal disc has been obtained. The level of expression for all the proteins has been quantitatively determined. The quantitative reproducibility of the analysis system has been estimated and all the controls studied as database reference to interpret the results of experiments with mutant discs. One example, corresponding to iro ¹ mutation, has been used to show how some of the changes observed with mutant discs clearly extend out of the limits defined by the controls. This enables us to generate comparative parameters for the study of proliferation, morphogenesis and differentiation of Drosophila and opens the possibility of rapidly defining the nature and quantity of changes in patterns of gene expression in developmental genetic studies. Received: 21 June 1996 / Accepted: 27 September 1996     

Characterization of the Tribolium Deformed ortholog and its ability to directly regulate Deformed target genes in the rescue of a Drosophila Deformed null mutant

 We have analyzed the Tribolium castaneum ortholog of the Drosophila homeotic gene Deformed (Dfd) and determined its expression pattern during embryogenesis in this beetle. Tc Deformed (Tc Dfd) is expressed in the blastoderm and the condensing germ rudiment in a region that gives rise to gnathal segments. During germ band extension Tc Dfd is expressed in the mandibular and maxillary segments, their appendages, and the dorsal ridge. Comparison of insect Dfd protein sequences reveals several highly conserved regions. To determine whether common molecular features reflect conserved regulatory functions we used the Gal4 system to express the Tribolium protein in Drosophila embryos. When Tc Dfd is expressed throughout embryonic ectoderm under the control of P69B, the beetle protein autoregulates the endogenous Dfd gene. In addition, the Drosophila proboscipedia gene (a normal target of Dfd) is ectopically activated in the antennal and thoracic segments. We also compared the ability of the beetle and fly proteins to rescue defects in Dfd ^– mutants by expressing each throughout the embryonic during embryogenesis. Both proteins rescued Dfd ^– defects to the same extent in that they each restore the development of mouth hooks and cirri, as well as cause gain-of-function abnormalities of posterior mouth parts. As before, pb was ectopically activated in the antennal segment. This is the first demonstration of the ability of a heterologous homeotic selector protein to directly regulate a target gene independent of an endogenous Drosophila autoregulatory loop. Received: 11 December 1998 / Accepted: 8 March 1999     

Rapid preparation of a panel of polyclonal antibodies to Drosophila segmentation proteins

 We describe a method for rapidly raising a panel of high quality polyclonal antibodies from bacterially expressed proteins. Approximately 1²/₃ days of preparation is required per protein. One step that speeds up the procedure is the visualization of purified bands by precipitated sodium dodecyl sulfate (SDS). Antigenicity of the purified recombinant proteins may be increased by precipitation in double-distilled water. The results of using the serums obtained for fluorescent staining of Drosophila embryos are shown. Received: 2 February 1998 / Accepted: 19 February 1998     

ribbon, raw, and zipper have distinct functions in reshaping the Drosophila cytoskeleton

rib and raw mutations prevent cells in a number of tissues from assuming specialized shapes, resulting in abnormal tubular epithelia and failure of morphogenetic movements such as dorsal closure. Mutations of zip, which encodes the nonmuscle myosin heavy chain, suppress the phenotypes of rib and raw, suggesting that rib and raw are not directly required for myosin function. Abnormal formation of the actin cytoskeletal structures underlying embryonic cuticular hairs suggests possible roles for rib and raw in organizing the actin cytoskeleton. The actin prehair structures are absent in rib mutants and abnormally shaped in raw mutants, indicating that the two genes have different functions required for organizing the actin cytoskeleton. Received: 4 December 1998 / Accepted: 26 January 1999     

An Arabidopsis homologue of the Drosophila meiotic gene Pelota

We have identified a plant homologue of the Drosophila meiotic gene Pelota in Arabidopsis thaliana (AtPelota1). This gene maps to chromosome 4 of Ara- bidopsis and is one of two Pelota homologues present in this plant. When the expression pattern of AtPelota1 was examined it was found to be expressed at similar levels in all plant tissues tested (whole plant, bud, stem, leaf and root). This expression pattern corresponds to that seen for some other Arabidopsis meiotic genes and their homologues. A search of the databases reveal that the AtPelota gene family is widespread with homologues present in higher and lower eukaryotes and archaebacteria, but not eubacteria. Received: 13 December 1999 / Accepted: 27 December 1999     

Rethinking cytoskeleton in plant reproduction: toward a biotechnological future?

 Sexual reproduction in plants is intimately connected to the activity of the cytoskeletal apparatus in reproductive cells. Because of the ease with which the pollen tube can be studied, it has become a model for studying many aspects of cell physiology related to the cytoskeleton, such as movement of organelles and vesicles and cell division. However, information about cytoskeletal proteins is still insufficient for determining cytoskeletal functions during reproduction, especially in terms of cell-cell interactions. One reason may be that cytological and biochemical research on the cytoskeleton of pollen and the embryo sac has not been complemented by sufficient research activity at genetic and molecular levels, and few laboratories are currently involved in this work. This might be because of problems in identifying appropriate applied applications of the work that might attract more investigation. Received: 20 January 1999/ Revision accepted: 29 March 1999