The small RNA profile during Drosophila melanogaster development

Small RNAs ranging in size between 20 and 30 nucleotides are involved in different types of regulation of gene expression including mRNA degradation, translational repression, and chromatin modification. Here we describe the small RNA profile of Drosophila melanogaster as a function of development. We have cloned and sequenced over 4000 small RNAs, 560 of which have the characteristics of RNase III cleavage products. A nonredundant set of 62 miRNAs was identified. We also isolated 178 repeat-associated small interfering RNAs (rasiRNAs), which are cognate to transposable elements, satellite and microsatellite DNA, and Suppressor of Stellate repeats, suggesting that small RNAs participate in defining chromatin structure. rasiRNAs are most abundant in testes and early embryos, where regulation of transposon activity is critical and dramatic changes in heterochromatin structure occur.     

Study of catheptic and acid phosphatase activities during development and metamorphosis ofDrosophila melanogaster

Summary Developmental stages ofDrosophila melanogaster cultured at 22 ± 1° C were collected and tested for catheptic activity and acid phosphatase activity.It was found that catheptic activity was absent in the egg as well as in the first and second larval instars. The activity first appears in the third instar larva and reaches its peak 24 to 48 hr after puparium formation. It then decreases, at first gradually and at pupal stage 9 (120 to 144 hr after puparium formation) abruptly, reaching zero level just before the emergence of the imago.The pattern of acid phosphatase activity in different developmental stages was found to be broadly similar to that of catheptic activity in the larval and pupal stages. However, the acid phosphatase activity was found to be exceptionally high in the egg in contrast to the catheptic activity.The authors are grateful to Prof. Dr. R. Weber, Zoological Institute of the University of Bern, for constructive criticism of this paper.     

The timing of RNA synthesis for spermiogenesis in organ cultures ofDrosophila melanogaster testes

Summary A method for the organ culture ofDrosophila testes is described which supports the differentiation of primary spermatocytes through the meiotic divisions to elongating spermatids. Autoradiographic and inhibitor studies reveal no evidence for RNA synthesis by developing spermatids ofDrosophila melanogaster; most, if not all, of the RNA required for the differentiation and elongation of sperm is synthesized earlier in the primary spermatocytes. Primary spermatocytes will differentiate into elongating spermatids in organ culture, despite severe (96–98%) inhibition of³H-uridine incorporation into RNA effected by 50 g/ml 3-deoxyadenosine. Protein synthesis in spermatids continues to be active in the presence of 3-deoxyadenosine, but that in growing spermatocytes is severely inhibited.Supported by grant number AEC PA 150-6 from the Atomic Energy Commission, and by grant number HD 03015 from the National Institutes of Health.     

Patterns of protein synthesis in salivary glands ofDrosophila melanogaster during larval and prepupal development

Summary Patterns of protein synthesis in the salivary glands ofDrosophila melanogaster have been studied throughout late larval and prepupal development by pulse labelling the tissues with³⁵S-methionine. Specific changes to the pattern of proteins synthesized during development are found and the significance of these changes is discussed in view of the known changes in gene (puffing) activity which occur at the same times. We review the problem of salivary gland function in prepupalDrosophila.     

Intercellular junctions during development and in tissue cultures ofDrosophila melanogaster: An electron-microscopic study

Summary The present investigation analyzes intercellular junctions in tissues with different developmental capacities. The distribution of junctions was studied inDrosophila embryos, in imaginal disks, and in cultures of disk cells that were no longer able to differentiate any specific pattern of the adult epidermis.The first junctions —primitive desmosomes andclose membrane appositions — already appear in blastoderm.Gap junctions are first detected in early gastrulae and later become more and more frequent.Zonulae adhaerentes are formed around 6 h after fertilization, whileseptate junctions appear in the ectoderm of 10-h-old embryos.Inwing disks of all stages studied (22–120 h), three types of junctions are found: zonulae adhaereentes, gap junctions, and septate junctions. Gap junctions, which are rare and small at 22 h, increase in number and size during larval development. The other types of junctions are found between all cells of a wing disk throughout development.All types of junctions that are found in normal wing disks are also present in theimaginal disk tissues cultured in vivo for some 15 years and in thevesicles of imaginal disk cells grown in embryonic primary cultures in vitro. However, gap junctions are smaller and in the vesicles less frequent than in wing disks of mature larvae.Thus gap junctions, which allow small molecules to pass between the cells they connect, are present in the early embryo, when the first developmental decisions take place, and in all imaginal disk tissues studied, irrespective of whether or not these are capable of forming normal patterns.     

Adipose tissue ofDrosophila melanogaster

Summary Using transplantation techniques it was shown that immature and supposedly mature stages of fat body from the larvae ofDrosophila melanogaster do not lyse rapidly in the lytic, internal environment of the young adult. In the younger tissue the protein granules (probably lysosomes) were just beginning to form, whereas in the older tissue the protein granules were at a maximum level. In both cases the implanted tissues became steadily smaller independently of the environment. The decrease in implant size was interreted to mean that the cells were lysing, since the average cell size did not change, and since many cells appear cytologically degenerate. However, the estimated rate of cell loss was much slower than in the case where the cells pass through metamorphosis. Some of the immature cells produced relatively high amounts of protein granules independently of the environment. Although the protein granules are at a maximum amount in both stages, it would appear that additional development must be required for the cells to become susceptible to the lytic environment of the young adult.On leave of absence for 1972–1973: Department of Genetics, University of Nijmegen, The Netherlands.The author expresses gratitude to the National Science Foundation for financial support (Research Grant GB 12969) and to Mrs. Barbara LaTendresse for her expert technical assistance.     

Rates of RNA synthesis during early embryogenesis in Drosophila melanogaster

We determined the absolute rates of RNA synthesis during embryogenesis in Drosophila melanogaster by measuring the incorporation of ³H-5-orotic acid into RNA, and the specific activity of the UTP pool. Initially (preblastoderm) the rate of RNA synthesis is relatively high, but declines to a lower level by gastrulation. The data suggest that RNA synthesis is initiated during very early embryogenesis.     

The three-dimensional optomotor torque system ofDrosophila melanogaster

Summary The well known optomotor yaw torque response in flies is part of a 3-dimensional system. Optomotor responses around the longitudinal and transversal body axes (roll and pitch) with strinkingly similar properties to the optomotor yaw response are described here forDrosophila melanogaster. Stimulated by visual motion from a striped drum rotating around an axis aligned with the measuring axis, a fly responds with torque of the same polarity as that of the rotation of the pattern. In this stimulus situation the optomotor responses for yaw, pitch and roll torque have about the same amplitudes and dynamic properties (Fig. 2). Pronounced negative responses are measured with periodic gratings of low pattern wavelengths due to geometrical interference (Fig. 3). The responses depend upon the contrast frequency rather than the angular velocity of the pattern (Fig. 4). Like the optomotor yaw response, roll and pitch responses can be elicited by small field motion in most parts of the visual field; only for motion below and behind the fly roll and pitch responses have low sensitivity.The mutantoptomotor-blind ^H31 (omb ^H31) in which the giant neurones of the lobula plate are missing or severely reduced, is impaired in all 3 optomotor torque responses (Fig. 5) whereas other visual responses like the optomotor lift/thrust response and the landing response (elicited by horizontal front-to-back motion) are not affected (Heisenberg et al. 1978).We propose that the lobula plate giant neurons mediate optomotor torque responses and that the VS-cells in particular are involved in roll and pitch but not in lift/thrust control. This hypothesis accommodates various electrophysiological and anatomical observations about these neurons in large flies.Abbreviation EMD elementary movement detector     

Rudimentary mutants ofDrosophila melanogaster

Summary The X-linkedrudimentary (r) mutants ofDrosophila melanogaster are pyrimidine auxotrophs and require exogenous pyrimidines (Nørby, 1970; Falk, 1976). We have established a set ofrudimentary cell lines that are derived from embryos, homozygous for eitherr ¹ orr ³⁶. The enzymatic activities of the pyrimidine synthesizing enzymes were measured in the mutant lines. We have further investigated the nutritional requirements of the mutant cells in vitro by using a pyrimidine free culture medium.Ther ¹ cell lines were found to express 3–7%dihydroorotase (DHOase) activity as compared to a wildtype cell line. Reducedaspartate transcarbamylase (ATCase) activity was measured in somer ¹ cell lines whereas wildtypecarbamylphosphate synthetase (CPSase) activity is expressed in allr ¹ cell lines. Ther ³⁶ cell line expresses wildtype activity ofDHOase andCPSase. ATCase activity was found to be reduced to 10% of the wildtype activity.The mutant cell lines do not proliferate in pyrimidine free minimal medium and cell proliferation is obtained by the addition of crude RNA. Proliferation of ther ¹ cells is restored by the supplementation of the minimal medium withdihydroorotate whereas proliferation of ther ³⁶ cells is restored by supplementation with eitherdihydroorotate orcarbamylaspartate.The results demonstrate that therudimentary phenotypesr ¹ andr ³⁶ are expressed at the cellular level and that the two mutant cell types behave as cellular pyrimidine auxotrophs in vitro.     

The development of ommatidial patterning in metamorphosed eye imaginal discs implants ofDrosophila melanogaster

Summary The development of the rhabdomeric pattern in the compound eye ofDrosophila has been studied using combined transplantation and electron microscope techniques. In a first series of experiments eye imaginal discs of increasing age were implanted into larvae ready to pupate, thus losing variable amounts of the normal time for development. A sequence of differentiative abilities was found in the metamorphosed test pieces. As far as the photoreceptor cells are concerned, the most prominent steps of this sequence are: ability to form groups with other similar elements, anatomical polarization of microvilli, establishment of the rhabdomeric pattern and formation of an equator line. The stability of determination of the equator line was tested in a second experimental series. Fragment of different topographical origin within the mature eye anlage were brought to metamorphosis by implantation into larvae ready to pupate. It was found that an equator line differentiates only in those pieces which according to the published anlage maps contain the prospective equator region prior to metamorphosis. The mitotic abilities of implanted eye imaginal discs were investigated by means of in vitro³H-thymidine pulse-labelling and light microscope autoradiography of the differentiated test pieces. During the third larval stage the eye anlage is traversed by two consecutive mitotic waves, each one of them producing different categories of receptor cells. The first, anterior wave predominantly produces cells oriented toward the poles of the eye within the ommatidia, while the second, posterior wave gives rise to elements exclusively in an equatorial position. The dynamics of this proliferation are discussed in relation to the findings in the implantation experiments. Silver-grain counts support the possibility that at least two successive cell divisions occur in the eye anlage between labeling with tritiated thymidine and beginning of morphological differentiation. The relevance of this finding for the understanding of the concept of acquisition of competence is discussed.     

The fate ofSepia in small populations ofDrosophila melanogaster

Asepia gene found inD. melanogaster collected in North Carolina, and wildtype flies from North Carolina, Bogotá, Barcelona, and California were used to strt 120 cultures that were maintained by mass transfers of adults every third week for more than a year. The frequency ofsepia was determined in these cultures at the termination of the experiment. Thesepia gene was present in considerable frequency (16%–65%) in all backgrounds except one; in cultures involving wildtype chromosomes from North Carolina, it was virtually eliminated. Each of the wildtype backgrounds exhibited a characteristic final frequency ofsepia, suggesting that they had reached at least quasi-stable equilibria. Although it is likely that the retention ofsepia depended upon the superiority of flies heterozygous for this mutant, the technique does not reveal whethersepia itself was involved in the apparent heterosis.     

The ultrastructure of the developing leg ofDrosophila melanogaster

Summary The ultrastructure of the imaginal discs ofDrosophila melanogaster was compared with that of other chitogenous tissues with different developmental capacities, namely, embryonic, larval, pupal and adult epidermis. Attention was paid to features which might be correlated with specific morphogenetic activities. Previous morphological studies of imaginal discs of Diptera were analyzed in detail and a somewhat revised view of imaginal disc structure emerged. The results reveal that the imaginal discs ofD. melanogaster consist of three types of cells: cells of the single layered disc epithelium, adepithelial cells and nerves. Four types of specialized junctions connect the cells of the disc epithelium: zonulae adhaerens, septate desmosomes, gap junctions and cytoplasmic bridges. The junctions are discussed in relation to their possible roles in adhesion and intercellular communication. It was concluded that gap junctions may be a more likely site for the intercellular communication involved in pattern formation than septate desmosomes. Evidence is presented that adepithelial cells are the precursors of imaginal muscles and that some cell lines (atelotypic) are in fact lines of adepithelial cells which can differentiate into muscle.Specific imaginal discs can be easily recognized by their overall morphology, i.e. patterns of folds. However, no ultrastructural features were found which we could correlate with the state of determination of the cells. Most differences in the ultrastructure of different discs at several developmental stages were attributable to different phases of cuticle secretion. The cells of the imaginal disc epithelium are packed with ribosomes but very little rough ER. The amount of rough ER increases rapidly at puparium formation. Cuticulin is recognizable 4–6 hours after puparium formation. Six hours after puparium formation, the cells of the disc epithelium are secreting the epicuticle of the pupa. As the imaginal disc of a leg everts from a folded sac to the tubular pupal leg, the cells of the disc epithelium change from tall columnar to cuboidal. A loss of microtubules in the long axis of the columnar cells accompanies this change. Prepupal morphogenesis of the leg appears to be caused by the change in cell shape. Evidence is presented which is incompatible with previous explanations of the mechanism of eversion of imaginal discs.There is some turnover of the cells of the disc epithelium as evidenced by autophagy and the occasional heterophagy of a dead neighbor. However this does not appear to be an important factor in the morphogenesis of discs. Plant peroxidase which was used as a tracer of proteins in the blood was taken up from the hemolymph by the disc epithelium. Imaginal disc cells contain many lipid droplets which coalesce and are replaced by glycogen during the prepupal period.

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Zusammenfassung Die Feinstrukturen der Imaginalscheiben, der embryonalen, larvalen, pupalen und adulten Epidermis, alles chitinbildende Gewebe, wurden untersucht und miteinander verglichen. Besondere Aufmerksamkeit legten wir auf ultrastrukturelle Merkmale, die mit spezifischen morphogenetischen Vorgängen korreliert sein können. Frühere Untersuchungen über die Morphologie der Imaginalscheiben bei Dipteren wurden kritisch analysiert und führten mit unseren Resultaten zu einem etwas veränderten Bild der Scheibenstruktur. Die Imaginalscheiben vonDrosophila melanogaster bestehen aus drei Zelltypen: Zellen des einschichtigen Epithels, adepitheliale Zellen und Nerven. Die Epithelzellen weisen vier spezialisierte Zellverbindungen auf: zonulae adherens, septate desmosomes, gap junctions und zytoplasmatische Brücken. Die Funktion dieser Zellverbindungen wird im Zusammenhang mit der Zelladhäsion und Zellkommunikation diskutiert. Es scheint, daß während der Musterbildung, die gap junctions, eher als die septate desmosomes, die Orte der Zellkommunikation sind. Wir haben gezeigt, daß adepitheliale Zellen Vorläufer der imaginalen Muskeln sind. Einige atelotypische Linien, die sich als Kulturen adepithelialer Zellen erwiesen, differenzieren Muskeln.Die Imaginalscheiben können leicht an ihrer Gesamtmorphologie, d.h. an ihrem Faltenmuster erkannt werden. Ultrastrukturelle Merkmale wurden jedoch nicht beobachtet, die mit dem Determinationszustand der Zelle korrelierbar wären. Während der Entwicklung sind die meisten Unterschiede in der Feinstruktur auf verschiedene Phasen der Kutikulasekretion zurückzuführen. Die Epithelzellen der Imaginalscheiben zeigen viele Ribosomen, besitzen aber nur sehr wenig endoplasmatisches Reticulum. Dieses nimmt erst bei der Pupariumbildung stark zu. 4–6 Std nach Puparisierung ist Kutikulin nachweisbar und nach 6 Std scheiden die Epithelzellen die Epikutikula aus. Während sich die Beinscheibe vom gefalteten Sack zum röhrenförmigen Bein ausstülpt, werden die länglichen Epithelzellen kubisch. Gleichzeitig mit dieser Formänderung verschwinden die Microtubuli in der Längsachse der Zellen. Die Morphogenese des Beines im Vorpuppenstadium scheint auf eine Änderung der Zellform zu beruhen. Früher beschriebene Erklärungen für den Mechanismus der Ausstülpung sind mit unseren Beobachtungen nicht vereinbar. Autophagozytose und gelegentlich Heterophagozytose einer toten Nachbarzelle konnten in den Epithelzellen nachgewiesen werden. Dies scheint jedoch kein wesentlicher Faktor für die Morphogenese der Scheibe zu sein. Pflanzenperoxydase, als Tracer-Protein im Blut, wird vom Scheibenepithel aus der Hämolymphe aufgenommen. Scheibenzellen enthalten viele Lipidtröpfchen, die sich vereinigen und während des Vorpuppenstadiums durch Glycogen ersetzt werden.

     

RNA polymerases during differentiation of avian erythrocytes

DNA-dependent RNA polymerase was analysed during the terminal differentiation stages of avian erythrocytes. It was found that the mature duck erythrocyte, although quiescent in RNA synthesis, contains clearly measurable quantities of RNA polymerase B (or II). Immature polychromatic erythrocytes, derived from anemic ducks and actively synthesizing hemoglobin mRNA, additionally contain significant amounts of RNA polymerase A (or I) and C (or III) previously not detected in these cells. These latter classes of enzymes, although present, are apparently not engaged in RNA synthesis in polychromatic erythrocytes.