Two orthodenticle-related genes in the short-germ beetle Tribolium castaneum

 To investigate the molecular basis of head evolution, we searched for genes related to the Drosophila orthodenticle (otd) homeobox gene in the short-germ beetle Tribolium castaneum. Unexpectedly, we found that there are two otd-related genes in Tribolium, with predicted homeodomains highly similar to that of the single Drosophila gene. One of the two genes (Tc otd-1) is more related in both amino acid sequence and expression pattern to fruitfly otd. Tc otd-1 is expressed in a broad anterior stripe in the blastoderm embryo, suggesting a role in early head segmentation similar to that of the Drosophila gene. The second gene (Tc otd-2) is more similar in sequence to the otd-related genes isolated from different vertebrate species (the Otx gene family). Tc otd-2 is not transcribed in the blastoderm, but is expressed later in more limited subsets of cells in the anterior brain. Both Tribolium genes and the Drosophila gene are, unlike the vertebrate genes, also expressed at the developing ventral midline of the embryo. Our results are consistent with the idea that an otd/Otx gene specified anterior head structures in the last ancestor common to arthropods and vertebrates. Within the arthropod lineage, we propose that this gene acquired a function in cells at the developing midline prior to the duplication that generated the two Tribolium genes. Received: 16 February 1996 / Accepted: 1 March 1996 Edited by C. Desplan     

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     

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     

A new family of adenylyl cyclase genes in the male germline of Drosophila melanogaster

We describe the cloning and characterization of a new gene family of adenylyl cyclase related genes in Drosophila. The five adenylyl cyclase-like genes that define this family are clearly distinct from previously known adenylyl cyclases. One member forms a unique locus on chromosome 3 whereas the other four members form a tightly clustered, tandemly repeated array on chromosome 2. The genes on chromosome 2 are transcribed in the male germline in a doublesex dependent manner and are expressed in postmitotic, meiotic, and early differentiating sperm. These genes therefore provide the first evidence for a role for the cAMP signaling pathway in Drosophila spermatogenesis. Expression from this locus is under the control of the always early, cannonball, meiosis arrest, and spermatocyte arrest genes that are required for the G₂/M transition of meiosis I during spermatogenesis, implying a mechanism for the coordination of differentiation and proliferation. Evidence is also provided for positive selection at the locus on chromosome 2 which suggests this gene family is actively evolving and may play a novel role in spermatogenesis. Received: 26 September 1999 / Accepted: 27 October 1999     

A system to efficiently maintain embryonic lethal mutations in the flour beetle Tribolium castaneum

 Due to its small size, short life cycle, and easy maintenance, the flour beetle Tribolium castaneum is well suited for the genetic analysis of development. One drawback of Tribolium as a genetic system is, however, the difficulty of keeping embryonic lethal lines. Presently, only few lethal mutations can be kept as balanced stocks. Therefore, heterozygous carriers must be identified anew in every generation in order to maintain a recessive embryonic mutation. To alleviate this problem we have devised a block system that allows the simultaneous processing of many mutant lines or test crosses for visual inspection of larval cuticle phenotypes. Using this technique, one person can maintain about 100 embryonic lethal stocks, which makes feasible the thorough genetic analysis of embryogenesis in this species. Received: 4 November 1998 / Accepted: 7 February 1999     

Changes in cell shape in the ventral neuroectoderm of Drosophila melanogaster depend on the activity of the achaete-scute complex genes

In the embryonic ventral neuroectoderm of Drosophila melanogaster the proneural genes achaete, scute, and lethal of scute are expressed in clusters of cells from which the neuroblasts delaminate in a stereotyped orthogonal array. Analyses of the ventral neuroectoderm before and during delamination of the first two populations of neuroblasts show that cells in all regions of proneural gene activity change their form prior to delamination. Furthermore, the form changes in the neuroectodermal cells of embryos lacking the achaete-scute complex, of embryos mutant for the neurogenic gene Delta, and of embryos overexpressing l’sc suggest that these genes are responsible for most of the morphological alterations observed. Received: 20 August 1999 / Accepted: 3 November 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     

Distinct functions of the Drosophila genes Serrate and Delta revealed by ectopic expression during wing development

 The Drosophila gene Serrate encodes a transmembrane protein with 14 epidermal growth factor-(EGF)-like repeats in its extracellular portion. It has been suggested to act as a signal in the developing wing from the dorsal side to induce the organising centre at the dorsal/ventral compartment boundary, which is required for growth and patterning of the wing. Ectopic expression of Serrate during wing development induces ectopic outgrowth of ventral wing tissue and the formation of an additional wing margin. Here we present data to suggest that both events are mediated by genes that are required for normal wing development, including Notch as receptor. In order for Serrate to elicit these responses the concomitant expression of wingless seems to be required. The lack of wings in flies devoid of Serrate function can be partially restored by Gal4-mediated expression of Serrate, whilst expression of wingless is not sufficient. Ectopic expression of Delta, which encodes a structurally very similar transmembrane protein with EGF-like repeats, provokes wing outgrowth and induction of a new margin under all conditions tested here, both on the dorsal and ventral side. Our data further suggest that Serrate can act as an activating ligand for the Notch receptor only under certain circumstances; it inhibits Notch function under other conditions. Received: 26 april 1996 / Accepted: 24 May 1996     

Patterning defects in the primary axonal scaffolds caused by the mutations of the extradenticle and homothorax genes in the embryonic Drosophila brain

During early brain development in Drosophila a highly stereotyped pattern of axonal scaffolds evolves by precise pioneering and selective fasciculation of neural fibers in the newly formed brain neuromeres. Using an axonal marker, Fasciclin II, we show that the activities of the extradenticle (exd) and homothorax (hth) genes are essential to this axonal patterning in the embryonic brain. Both genes are expressed in the developing brain neurons, including many of the tract founder cluster cells. Consistent with their expression profiles, mutations of exd and hth strongly perturb the primary axonal scaffolds. Furthermore, we show that mutations of exd and hth result in profound patterning defects of the developing brain at the molecular level including stimulation of the orthodenticle gene and suppression of the empty spiracles and cervical homeotic genes. In addition, expression of a Drosophila Pax6 gene, eyeless, is significantly suppressed in the mutants except for the most anterior region. These results reveal that, in addition to their homeotic regulatory functions in trunk development, exd and hth have important roles in patterning the developing brain through coordinately regulating various nuclear regulatory genes, and imply molecular commonalities between the developmental mechanisms of the brain and trunk segments, which were conventionally considered to be largely independent. Received: 4 October 1999 / Accepted: 10 January 2000     

Growth, metastasis, and invasiveness of Drosophila tumors caused by mutations in specific tumor suppressor genes

 More than 50 genes have been identified in Drosophila by loss-of-function mutations that lead to overgrowth of specific tissues. Loss-of-function mutations in the lethal giant larvae, discs large, or brain tumor genes cause neoplastic overgrowth of larval brains and imaginal discs. In the present study, the growth and metastatic potential of tumors resulting from mutations in these genes were quantified. Overgrown brains and imaginal discs were transplanted into adults and β-galactosidase accumulation was used as a marker to identify donor cells. Mutations in these three genes generated tumors with similar metastatic patterns. For brain tumors, the metastatic index (a measure we defined as the fraction of hosts that acquired secondary tumors normalized for the amount of primary tumor growth) of each of the three mutants was similar. Analysis of cell proliferation in mutant brains suggests that the tumors arise from a population of several hundred cells which represent only 1–2% of the cells in third instar larval brains. For imaginal disc tumors from lethal giant larvae and brain tumor mutants, it is shown for the first time that they can be metastatic and invasive. Primary imaginal disc tumors from lethal giant larvae and brain tumor mutants formed secondary tumors in 43 and 53% of the hosts, respectively, although the secondary tumors were, in general, smaller than the secondary tumors derived from primary brain tumors. Received: 18 August 1997 / Accepted: 16 October 1997     

Molecular Characterization and Evolution of the Amylase Multigene Family of Drosophila ananassae

Drosophila ananassae is known to produce numerous alpha-amylase variants. We have cloned seven different Amy genes in an African strain homozygous for the AMY1,2,3,4 electrophoretic pattern. These genes are organized as two main clusters: the first one contains three intronless copies on the 2L chromosome arm, two of which are tandemly arranged. The other cluster, on the 3L arm, contains two intron-bearing copies. The amylase variants AMY1 and AMY2 have been assigned to the intronless cluster, and AMY3 and AMY4 to the second one. The divergence of coding sequences between clusters is moderate (6.1% in amino acids), but the flanking regions are very different, which could explain their differential regulation. Within each cluster, coding and noncoding regions are conserved. Two very divergent genes were also cloned, both on chromosome 3L, but very distant from each other and from the other genes. One is the Amyrel homologous (41% divergent), the second one, Amyc1 (21.6% divergent) is unknown outside the D. ananassae subgroup. These two genes have unknown functions. Received: 30 May 2000 / Accepted: 17 July 2000