Range of potential functions of the Drosophila melanogaster hdc gene

The Drosophila melanogaster hdc gene controls trachea branching, which starts during embryo development. Expression in imaginal disks and reproductive organs suggests additional functions for the hdc gene. The gene was demonstrated to have a maternal effect, which was denied previously. Analysis of cell proliferation in imaginal disks with hdc mutations showed that the gene does not possess tumor suppressor properties at the levels of mosaic cuticle clones of adults and transplanted imaginal disks. Transplanted imaginal disks homozygous, but not heterozygous, for an hdc mutation were found to affect oogenesis in the recipient females, implicating the hdc activity in exchanging signals between different organs. Amino acid sequence analysis of the HDC protein revealed a region homologous to the human HRS proteins, which directly interact with the NF2 tumor suppressor on experimental evidence.     

Control of pupal commitment in the imaginal disks of Precis coenia (Lepidoptera: Nymphalidae)

When final (5th) instar larvae of Precis coenia were treated with the juvenile hormone analog (JHA) methoprene, they underwent a supernumerary larval molt, except for certain regions of their imaginal disks, which deposited a normal pupal cuticle. Evidently those regions had already become irreversibly committed to pupal development at the time JHA was applied. By applying JHA at successively later times in the instar, the progression of pupal commitment could be studied. Pupal commitment in the proboscis, antenna, eye, leg and wing imaginal disks occurred in disk-specific patterns. In each imaginal disk there were distinct initiation sites where pupal commitment began during the first few hours of the final larval instar, and from which commitment spread across the remainder of the disk over a 2- to 3-day period. The initiation sites were not always located in homologous regions of the various disks. As a rule, pupal commitment also spread from imaginal disk tissue to surrounding epidermal tissue. The regions of pupal commitment in all disks except those of the wings, coincided with the regions of growth of the disk. Only portions of the disk that had undergone cell division and growth underwent pupal commitment. Shortening the growth period did not prevent pupal commitment in the wing imaginal disk, indicating that, in this disk at least, a normal number of cell divisions was not crucial in reprogramming of disk cells for pupal cuticle synthesis. The apparent growth spurt of imaginal disks that occurs during the last part of the final larval instar is merely the final stage of normal and constant exponential growth. Juvenile hormone (JH) and ecdysteroids appeared to play little role in the regulation of normal imaginal disk growth. Instead, growth of the disks may be under intrinsic control. Interestingly, even though endogenous fluctuation in JH titers do not affect imaginal disk growth, exogenous JHA proved able to inhibit both pupal commitment, cell movement, and growth of the disks during the last larval instar. This function of JH could be important under certain adverse conditions, such as when metamorphosis is delayed in favor of a supernumerary larval molt.     

Genetic, cytogenetic and developmental analysis of the Drosophila melanogaster tumor suppressor gene lethal(2)tumorous imaginal discs (l(2)tid)

Three of the twenty recessive-lethal tumor suppressor genes of Drosophila cause imaginal disc tumors in the homozygously mutated state. One of these is the lethal(2)tumorous imaginal discs (l(2)tid) gene. Histological preparations show the tumorous imaginal disc epithelium to consist of a mosaic of cells in monolayer and cells in clumped arrangement. In contrast, the wild-type imaginal disc epithelium is comprised exclusively of cells in monolayer arrangement. Mutant imaginal disc tissue pieces implanted into ready-to-pupariate wild-type larvae fail to differentiate. Implantation of l(2)tid imaginal disc tissue pieces in vivo into wild-type adult flies revealed a lethal, tumorous growth comparable to that in situ, thus characterizing the l(2)tid imaginal discs as truly malignant. The phenotypes of double mutants between two l(2)tid alleles and tumor suppressor genes, such as lethal(2)giant larvae and lethal(2)brain tumor, and the epithelial overgrowth mutant lethal(2)fat are described and discussed. Finally, we present the genetic, cytogenetic and molecular localization of the l(2)tid gene to the giant chromosome bands 59F4-6.     

Notch signaling activates Yorkie non-cell autonomously in Drosophila

In Drosophila imaginal epithelia, cells mutant for the endocytic neoplastic tumor suppressor gene vps25 stimulate nearby untransformed cells to express Drosophila Inhibitor-of-Apoptosis-Protein-1 (DIAP-1), conferring resistance to apoptosis non-cell autonomously. Here, we show that the non-cell autonomous induction of DIAP-1 is mediated by Yorkie, the conserved downstream effector of Hippo signaling. The non-cell autonomous induction of Yorkie is due to Notch signaling from vps25 mutant cells. Moreover, activated Notch in normal cells is sufficient to induce non-cell autonomous Yorkie activity in wing imaginal discs. Our data identify a novel mechanism by which Notch promotes cell survival non-cell autonomously and by which neoplastic tumor cells generate a supportive microenvironment for tumor growth.     

Headcase gene--proliferation or hormonal control?

A new insertion allele of the hdc gene was isolated and described. The nucleotide sequence of the coding region had no detectable homology with genomic DNA of any other Drosophila species, except for D. mauritiana. Gene expression was found both in adult testes and ovaries and at embryonic and larval stages. This expression pattern exhibits a strong similarity to that of cell-cycle genes. In contrast to cycE (a typical cell-cycle gene), which leads to expression termination after in vivo culturing of the wing disk, hdc did not arrest expression. It is concluded that hdc is a species-specific differentiation gene, whose regulatory activity in the development of an organism differs from that of proliferation genes.     

Phases of the effect of the homeotic Walker mutation in the development of Drosophila melanogaster

In this work, critical ontogenetic stages for wing traits affected by temperature-sensitive mutation Walker (Wk) were determined. The interaction between the Wk gene and some genes responsible for the cell-cycle control was studied. At various ontogenetic stages, the mutants were exposed to 17 degrees C for 12 h, and, at the beginning of oviposition, the fly age was registered. Nine types of wing abnormalities were classified. The temperature treatment during three developmental stages (12-24, 48-60, and 96-108 h) resulted in a decrease in normal wing number and a substantial increase in wing abnormalities. Different morphological types of imaginal disks were revealed: nondifferentiated disks, those lacking the notum region, and those with duplications of wing-forming regions. The allele-specific interaction between Wk and allele v27 of the Klp61F gene was also revealed. We suggest that gene Wk is a high-ranking gene in the system of genetic control of ontogeny, because the Wk mutation is manifested in numerous phenotypic variants both in the control and in the experiment and a complete set of these variants was observed at each developmental stage upon temperature treatment. The pleiotropic effect of the Wk gene on the formation of some Drosophila organs, including eyes and halters which are beyond the scope of this report, is in agreement with this suggestion.     

The protein of the ejaculatory bulb (PEB) in Drosophila melanogaster. 2. The organ specificity of its expression

Tissue localization and synthesis of PEB (the major protein of ejaculatory bulb (EB) in D. melanogaster) were studied. PEB and its minor form hPEB were detected only in EB as shown by immunoblotting technique. Transplantation of genital imaginal disks and the EBs of young males into recipients of strain with different PEB electrophoretic mobility showed that synthesis and accumulation of PEB do not depend on EB interaction with other organs. In genotypic females transformed into males and intersexes by sex-reversing mutations PEB was detected in EB but not in other organs.     

The relationship of decapentaplegic and engrailed expression in Drosophila imaginal disks: do these genes mark the anterior-posterior compartment boundary?

Imaginal disks, the primordia of the adult appendages in Drosophila, are divided into anterior and posterior compartments. However, the developmental role of such compartments remains unclear. The expression of decapentaplegic (dpp), a pattern formation gene required for imaginal disk development, has the intriguing property of being expressed in a line at or near the boundary between these compartments. Here, we compare the distribution of dpp-driven reporter gene expression to the pattern of expression of the engrailed (en) gene, known to be required for the maintenance of the compartment boundary. Using confocal microscopy to obtain single cell resolution, we have determined that the majority of the en+ imaginal disk cells expressing the dpp-driven reporter genes about those cells expressing en, while a small percentage of dpp reporter gene expressing cells also express en. In posterior regions of en mutant disks, where compartmentalization is abnormal, we observe ectopic expression of the dpp-driven reporter genes. We conclude that the pattern of dpp expression in imaginal disks is delimited in part through the direct or indirect repression by engrailed. Our results lead us to question the widely held assumption that the anterior edge of en expression demarcates the A/P compartment boundary.     

Parameters influencing the acquisition of competence for metamorphosis in imaginal disks of Drosophila

When imaginal disks from first and early second instar larvae of Drosophila are transplanted into larval hosts that are ready to pupate, they are unable to differentiate adult structures. The disks gradually become competent to respond with imaginal differentiation towards the end of the second larval instar (Fig. 1). The first sign of imaginal differentiation is a light-orange pigment that appears in the presumptive eye region when eye-antennal disks from early second instar larvae were subjected to immediate metamorphosis. This pigment was identified as being composed of ommochromes and drosopterins.Incompetent eye-antennal disks from early second instar donors were cultured in adult females for 2 to 5 days, and then retransplanted into late third instar larval hosts. If the adult host flies were kept on standard food the disks grew by cell multiplication (Fig. 2c) and became competent to undergo imaginal differentiation (Fig. 3). If, on the other hand, the adult hosts were starved on a protein-free sugar diet, cell divisions were effectively blocked in the disks. These did not noticeably grow (Fig. 2b) and remained incompetent (Fig. 3). The block caused by starvation proved to be reversible. Based on these results the hypothesis is advanced that the acquisition of competence requires a minimum number of cell divisions to take place in the disk primordium.     

Role of the <Emphasis Type="Italic">porcupine</Emphasis> gene in the development of the wing imaginal disk of <Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

A search for the genes interacting with the Merlin tumor suppressor gene revealed a Merlin-porcupine interaction during wing morphogenesis. Ectopic expression of the porcupine gene in the wing imaginal disk reduced the adult wing, while addition of an UAS construct with a full-length or truncated copy of the Merlin gene partly restored the wing phenotype. The highest restoration level was observed upon adding the fragments coding for the C end of the Merlin protein. In addition, the porcupine gene was shown to mediate the wingless gene autoregulation, which occurs at two ontogenetic stages, segmentation during embryo development and determination of the wg expression band at the boundary between the dorsal and ventral compartments of the wing imaginal disk.     

Development of a detection system for histidine decarboxylating lactic acid bacteria based on DNA probes, PCR and activity test

On the basis of the comparison of the nucleotide sequences of the histidine decarboxylase genes ( hdc A) of Lactobacillus 30A and Clostridium perfringens and the amino acid sequences of these histidine decarboxylases and those of Lactobacillus buchneri and Micrococcus , oligonucleotides unique to the hdc A genes were synthesized and used in PCR. All histidine-decarboxylating lactic acid bacteria gave a signal with primer set JV16HC/JV17HC in PCR. In addition to this primer set, CL1/CL2 and CL1/JV17HC were also useful for the detection of histamine-forming Leuconostoc œnos strains in PCR. The 150 base pair amplification product of the decarboxylating Leuc. œnos strain generated with primer set CL1/CL2 was sequenced. Alignment studies showed a high degree of relatedness among the hdc A gene products of Gram-positive bacteria.
The amplification products of the hdc A genes from Lact. buchneri and Leuc. œnos were used to serve as a DNA probe in hybridization studies. All histidine-decarboxylating lactic acid bacteria gave a hybridization signal with the DNA probes. In hybridization only one false-positive signal with a Lactobacillus lindneri strain was observed, which was anticipated to contain a truncated hdc A gene.
In addition to these DNA probe tests, a simple and reliable activity test is presented, which can be used during starter selection to test strains for histidine decarboxylase activity.     

Untersuchungen zum Problem der Wachstumsregulation

Summary A growth-regulation-system is defined in mathematical terms in order to interpret the observed behavior of regenerating and growing imaginal disks of the wings inEphestia kühniella.It is assumed that the growth-rate of an imaginal disk depends upon two variables, a variablek which is interpreted as the concentration of the moulting hormone and a second variablez representing the size of the disk measured by the number of cells. A rising hormone concentration tends to increase and a rising number of cells tends to decrease the growth-rate of a disk. If two imaginal disks of different size are present in one larva and the concentration of the moulting hormone is the same for both, then the growth-rate of the smaller imaginal disk can only be greater than or equal to the growth-rate of the larger one. The model contains the possibility that a small imaginal disk grows independently of the hormone level with a maximum and constant rate.On the basis of that model some theorems are derived in order to simplify the interpretation of the empirical data which consist of daily counts of cell divisions on the upper side of the imaginal disks after successive extirpation or one, two of three imaginal disks. The data do not contradict the model and suggest that before reaching a critical size the regenerating imaginal disk grows independently of the hormone level while afterwards the growth-rate depends upon the two variables mentioned above.Some ideas are discussed about the control of the hormone level and the conditions under which the time for the regulation of size differences is minimized.

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Die Arbeit wurde von der Deutschen Forschungsgemeinschaft unterstützt. FrauHildegard Framenau und Herrn Dr.Eduard Amtmann danke ich für ihre Mitarbeit.     

Effect of mutations in <Emphasis Type="Italic">Drosophila melanogaster</Emphasis> tumor suppressor <Emphasis Type="Italic">Merlin</Emphasis> on proliferation and differentiation of wing cells

Experiments on transplantation of wing imaginal discs homozygous for a mutation in the tumor suppressor gene Merlin have demonstrated that this mutation does not induce malignant tumors. Marking of the wing disc compartment borders by specific antibodies showed the absence of essential compartment border defects in case of the Merlin mutation. Drosophila melanogaster cells mutant for Merlin have shorter cell cycle than normal cells. Proliferation of imaginal discs lasts longer in case of the mutation. It is known that beginning from some moment of development, wing veins serve as clonal restriction lines that cannot be crossed by growing mosaic clones. We showed that the Merlin mutation leads to depression of vein clonal restriction property. This means that this gene is involved not only in the control of cell proliferation, but also in the control of cell mobility and adhesion.     

Decapentaplegic: a gene complex affecting morphogenesis in Drosophila melanogaster

The decapentaplegic gene complex (2-4.0) in Drosophila melanogaster is defined by a series of allelic mutations affecting imaginal disk development. Decapentaplegic (dpp) mutant individuals exhibit a variety of pattern deficiencies and duplications in structures derived from one or more of the 15 major imaginal disks. Based on dpp mutant phenotypes, we suggest that the dpp gene complex is involved in the elaboration of positional information within developing epidermal tissue. The dpp mutations are recessive and fall into six phenotypic classes. Milder alleles (classes I and II) affect only one or a few disks while most alleles (classes III, IV, V and EL) affect all major imaginal disks. Class EL homozygotes are embryonic lethals; development is arrested before germ-band shortening late in gastrulation. Presently inseparable from EL, is a haplo-insufficient function (Hin-d) associated with the distal (left) end of the dpp gene complex. The dpp gene complex occupies most or all of 22F1--3, three densely staining polytene chromosome bands. A colinearity exists between map positions of the four identified functional units within the complex and the severities of mutant phenotypes caused by disruption of these functions. Most dpp mutations are gross chromosomal rearrangements; they exert polar effects on the decapentaplegic functions that are proximal to the rearrangement breakpoints in 22F. Many structural similarities exist between the decapentaplegic and bithorax gene complexes.     

Microtubules and tracheole migration in wing disks of Galleria mellonella

Tracheoles migrate into the lacunae of wing imaginal disks that are cultured in vitro in medium containing α-ecdysone. Colchicine and vinblastin prevented tracheole migration in vitro under conditions which did not affect the viability of the disks. Cytochalasin B also inhibited tracheole migration, but only at concentrations which were deleterious to the disks. Our ultrastructure findings were in accord with the hypothesis that microtubules, but perhaps not microfilaments, are required for tracheole migration.     

Genetic control of mitosis. Adaptive modifications of v158 mutation expression

Cytogenetic parameters of mitosis were studied in the neural ganglions and imaginal disks of the third-instar Drosophila larvae of the marker lines ry506 and w; Cy/L; D/Sb; two wild-type lines Lausenne and Hikone-AW; and the v158 line mutant for the cell-cycle gene in the 85F locus. The control lines and their various tissues differ in a number of mitotic traits, which are believed to be the natural modifications of chromosome condensation and segregation and do not disturb homeostasis of the developing ry506, Lausenne, and Hikone-AW flies. Mutation v158 affects centromere disjunction. In imaginal disks, this results in arrest of either mitosis or anaphase initiation, whereas, in the neural ganglions, chromosomes integrate into a monopolar spindle at prophase and unipolar cells appear in anaphase. Different effects of the mutation in various tissues are assumed to be caused by different activity of the checkpoint system. When the mutation was maintained heterozygous for a long time, adaptive modification of its expression was observed. A comparison of the rates of two major and parallel mitotic processes, spindle formation and chromosome condensation, showed that adaptive modification can proceed via the adjustment of these rates.     

Drawing a Stripe in Drosophila Imaginal Disks: Negative Regulation of Decapentaplegic and Patched Expression by Engrailed

During development of the Drosophila adult appendage precursors, the larval imaginal disks, the decapentaplegic (dpp) gene is expressed in a stripe just anterior to the anterior/posterior (A/P) compartment boundary. Here, we investigate the genetic controls that lead to production of this stripe. We extend previous observations on leaky engrailed (en) mutations by showing that mutant clones completely lacking both en and invected (inv) activity ectopically express dpp-lacZ reporter genes in the posterior compartment, where dpp activity ordinarily is repressed. Similarly, patched (ptc) is also ectopically expressed in such posterior compartment en(-)inv(-) null clones. In contrast, these en(-)inv(-) clones exhibit loss of hedgehog (hh) expression. We suggest that the absence of dpp expression in the posterior compartment is due to direct repression by en. Ubiquitious expression of en in imaginal disks, produced by a hs-en construct, eliminates the expression of dpp-lacZ in its normal A/P boundary stripe. We identify three in vitro Engrailed binding sites in one of our dpp-lacZ reporter gene. Mutagenesis of these Engrailed binding sites results in ectopic expression of this reporter gene, but does not alter the normal stripe of expression at the A/P boundary. We propose that the en-hh-ptc regulatory loop that is responsible for segmental expression of wingless in the embryo is reutilized in imaginal disks to create a stripe of dpp expression along the A/P compartment boundary.