Histotypic reaggregation of dissociated imaginal disc cells ofDrosophila melanogaster culturedin vivo

Summary Cell dissociates of wing and leg imaginal discs perform, culturedin vivo, disc specific morphogenetic movements leading to their aggregation into layers and vesicles. These histological patterns correspond directly with the final cuticular patterns which appear after metamorphosis of the same implants.The reaggregation into layers is achieved before the entry of the blastems into metamorphosis, and reflects the existence of traits of differentiation in the isolated imaginal disc cells.The bearing of selective affinity and reaggregation on cell differentiation of imaginal discs cells is discussed.Supported by a grant of the Juan March Foundation.     

Chitin synthesis in Spodoptera frugiperda wing imaginal discs. III: Role of the peripodial membrane

We determined the contribution of the peripodial membrane to chitin synthesis in cultured wing imaginal discs of Spodoptera frugiperda. This was accomplished by examining chitin synthesis in vitro in intact imaginal discs, in the peripodial membrane, and in imaginal discs in which the peripodial membrane had been injured. Chitin synthesis in peripodial membrane-deprived imaginal discs, peripodial membrane injured imaginal discs, and peripodial membrane fragments was assessed by measuring incorporation of [¹⁴C]GlcNAc after treatment with 20-hydroxyecdysone in tissue culture. Removing or injuring the peripodial membrane resulted in a marked decrease in ecdysteroid-dependent chitin synthesis in these wing discs compared with intact wing discs. In addition, a break in the ecdysteroid treatment of 4 h reduced chitin synthesis in the wing discs substantially. These biochemical experiments were supplemented with ultrastructural and immunocytochemical approaches. A wheat germ agglutinin colloidal gold complex was used to visualize the presence of chitin synthesized by wing discs including the peripodial membrane. These experiments confirmed the importance of an intact peripodial membrane for optimal production of cuticle by the wing pouch. Our results demonstrate that for opti-ma1 production of chitin in tissue culture, wing discs must be treated with 20-hydroxyecdysone for an uninterrupted period of 48 h, and the peripodial membrane of these imaginal discs must be present and uninjured. © 1995 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

In vitro culture of Drosophila imaginal disc cells: aggregation, sorting out, and differentiative abilities

This paper describes the aggregation in vitro of cells dissociated from imaginal discs and demonstrates the sorting out of undifferentiated cells from different imaginal discs and from differently determined regions of the same imaginal disc, as well as the abilities of such cells to undergo pattern reconstruction when injected into larvae. Dissociated cells begin to aggregate by 1.5 hr of rotation. By 5 hr of rotation, large aggregates of loosely associated cells appear. By 18 hr the aggregates have condensed and taken on a characteristic epithelial structure. To study sorting out in undifferentiated cells, we combined a histochemical stain for acid phosphatase with the use of the acid phosphatase null mutant acphn-11. We performed cell mixing experiments with 0-2 (prospective notum) and 2-8 (prospective wing) fragments, with the A and P (prospective anterior and posterior) fragments of the dorsal mesothoracic disc and with mixtures of cells from ventral prothoracic and dorsal mesothoracic discs. We found that prospective anterior and posterior dorsal mesothoracic cells do not sort out, but that prospective notum and wing and leg and wing cells do. The results from differentiated implants are consistent with those from undifferentiated mixes.     

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.

     

Homologies of positional information in thoracic imaginal discs ofDrosophila melanogaster

Summary The regulative behavior of fragments of the imaginal discs of the wing and first leg was studied when these fragments were combined with fragments of other thoracic imaginal discs. A fragment of the wing disc which does not normally regenerate when cultured could be stimulated to regenerate by combination with certain fragments of the haltere disc. When combined with a haltere disc fragment thought to be homologous by the criteria of morphology and the pattern of homoeotic transformation, such stimulated intercalary regeneration was not observed. Combinations of first and second leg disc fragments showed that a lateral first leg fragment could be stimulated to regenerate medial structures when combined with a medial second leg disc fragment but not when combined with a lateral second leg disc fragment. Combinations of wing and second leg disc fragments showed that one fragment of the second leg disc is capable of stimulating regeneration from a wing disc fragment while another second leg disc fragment fails to stimulate such regeneration. It is suggested that absence of intercalary regeneration in combinations of fragments of different thoracic imaginal discs is a result of homology or identity of the positional information residing in the cells of the fragments. The pattern of correspondence of positional information revealed by this analysis is consistant with the pattern of homology determined by morphological observation and by analysis of the positional specificity of homoeotic transformation among serially homologous appendages. The implications of the existence of homologous positional information in wing and second leg discs which share a common cell lineage early in development are discussed.     

Integument and sensory nerve differentiation ofDrosophila leg and wing imaginal discs in vitro

Summary An ultrastructural analysis is presented of the cuticular and neural structures formed by the prothoracic leg and wing imaginal discs of maleDrosophila melanogaster larvae during culture in vitro with 0.2 g/ml of -ecdysone. A pupal cuticle, and subsequently an imaginal cuticle with a well-defined epicuticle and a laminated endocuticle is formed. The ultrastructure of the epidermis and of cuticular structures such as bristles, trichomes, apodemes, and tracheoles is very similar to that found in situ. Dendrites and nerve cell bodies are formed in vitro, and sensory axons form nerve bundles similar to those of normal appendages in situ, despite their isolation from the central nervous system. It is concluded that at the ultrastructural level, differentiation in vitro closely parallels the normal course of development.     

Fusion ofDrosophila eye-antennal imaginal discs during differentiation in vitro

Summary Pairs of eye-antennal discs, attached to the cephalic ganglia, were cultured in vitro with a concentration of -ecdysone optimal for imaginal differentiation. The eye-antennal discs fused to form a vesicle inside which the antennae were partially everted, and on the inner surface of which imaginal structures differentiated. The epithelium of the discs was continuous, and an integrated pattern of bristles and hairs differentiated in vitro. In particular, the median ocellus, a unified structure derived partially from each disc, differentiated normally.     

The initiation of somatic embryos and adventitious roots from developing zygotic embryo explants of Cercis canadensis L. cultured in vitro

Zygotic embryo explants of Cercis canadensis L. cultured in vitro responded to 1 and 5 M 2,4-dichlorophenoxyacetic acid (2,4-D) or 50 M -naphthaleneacetic acid (NAA) by initiating somatic embryos and adventitious roots. Somatic embryos and adventitous roots were formed from developing zygotic embryos, while fully developed embryos collected from mature seed initiated only adventitious roots. Following 2,4-D application, the number of somatic embryos decreased while adventitious roots increased with increasing developmental age of the explant. The greatest number of somatic embryos were initiated with a 5 to 20 day exposure to 5 M 2,4-D from zygotic embryos collected between 75 and 82 days post-anthesis in 1987. Somatic embryos formed directly from epidermal and subepidermal cells, while adventitious roots developed from interior cortex cells. Normal somatic embryos were recovered after a 20 day exposure to 5 M 2,4-D and acclimated to greenhouse conditions.     

Specific synaptic systems in reaggregated spherules from dissociated chick cerebellum cultivated in vitro

Summary Studies were performed on spherules of approximately 100–300 m in diameter obtained from in vitro cultures of reaggregates of embryonic fragments of cerebellum from 10–12 day-incubated chick embryos, dissociated with trypsin and cultivated in a rotating shaker for a maximum of 21 days. The differentiated neurons within these spherules included a few Purkinje cells, many granule cells and type II Golgi cells, as well as many glial cells. Zones rich in synaptic knobs and other simple synaptic structures as well as complex synaptic systems with numerous active points of contact, were visible in various parts of the spherule. Typical glomeruli consisting of a varicosity or rosette joined to the dendritic claws of the granule cells, and en marron systems with perikarya of type II Golgi cells were easily recognised. The complete absence of extracerebellar afferents confirms that both the granule and Golgi cells are capable of making synaptic connections with afferents different from those normally formed by extracerebellar mossy or climbing fibres.The experimental findings confirm that the recipient neurons determine the specific synaptic pattern regardless of the nature of the afferents, and furthermore demonstrate that the clinging activity of the recipient neuron determines the synaptogenic behaviour of nervous pathways.This work was supported by a grant from the Consiglio Nazionale delle Ricerche. Technical assistance was given by D. Scorsini for the ultrathin sectioning and electron microscopy, and by G. Gentili for the in vitro preparation and micrographs     

Chironomus tentans epithelial cell lines sensitive to ecdysteroids, juvenile hormone, insulin and heat shock

A new culture medium, ZW, and the preparation of an extract of adult Drosophila, FX, are described, which for the first time allow the in vitro proliferation of normal Drosophila cells in the absence of undefined heterologous components. Cells from 6-hour-old Drosophila embryos can extensively differentiate and/or proliferate in ZW supplemented with FX and insulin. Cells isolated from wing discs of 90–120-hour-old larvae require ecdysterone for proliferation in ZW, in addition to FX and insulin. Explanted ovaries, testes, genital discs and intact or halved wing discs of 100-hour-old larvae grow in the same medium, at least in part due to cell proliferation. High concentrations of ecdysterone prevent differentiation and/or proliferation of cells from embryos and from wing discs and cause the lysis of most isolated imaginal disc cells grown in vitro, while cuticular differentiations are induced in wing discs and disc fragments grown in vitro.     

Gap junctions are non-randomly distributed inDrosophila wing discs

Summary The distribution of gap junctions in mature larvalDrosophila melanogaster wing discs was analyzed by means of quantitative electron microscopy. Gap junctions are non-randomly distributed in the proximal-distal disc axis and in the apical-basal cell axis of the epithelium. In the epithelial cells, the surface density, number and length of gap junctions are greatest in the apical cell region and distal disc region. The average gap junction surface density is 0.0572 m^–1 and 2.77% of the lateral cell surface is composed of gap junctions. In the adepithelial cells, the gap junction surface density is 0.0005 m^–1 and 0.06% of the cell surface is composed of gap junctions. No gap junctions were observed between epithelial cells and adepithelial cells. The absolute area of gap junctions was estimated in a proximal-distal strip of cells in the disc and is considerably less in the folded regions of the epithelium compared to the flat notum and wing pouch regions. The results are discussed with respect to pattern formation and growth control in imaginal discs.     

Aldehyde oxidase distribution in the imaginal discs of some diptera

Summary In the imaginal discs ofMusca domestica, Drosophila melanogaster, D. simulans, D. hydei, andZaprionus spec. the enzyme aldehyde oxidase (AO) appeared in a clear-cut pattern. In the leg and eye-antennal discs of these species this pattern shows a high degree of conformity, while that of the wing and haltere discs is species-specific.No aldehyde oxidase activity was detected in the imaginal discs ofCalliphora erythrocephala, Phormia regina orLucilia cuprina, but the discs of these species are characterized by grossly similar patterns of 5-nucleotidase. Since the other species studied lack this enzyme, the two enzymes may perform similar functions in the morphogenesis of the discs.The coincidence of the sharp boundary of the AO pattern in the leg and wing discs ofD. melanogaster with the boundary between the anterior and posterior disc compartments gives a strong indication for the existence of analogous compartments in other discs showing a similar sharply bounded AO pattern. Compartmentalization may be considered a general phenomenon which occurs in discs of all segments and is not restricted toD. melanogaster. From the changes in the AO pattern during disc development it can be deduced that the localisation of this enzyme is regulated by supracellular determination involving positional information.     

α-Isopropylmalate synthase from Alcaligenes eutrophus H 16

The purified isopropylmalate synthase of Alcaligenes eutrophus H 16 reacted with the following -keto acids and acyl-coenzyme A derivatives (in the sequence of decreasing affinities): -ketoisovalerate, -keto-n-valerate, -ketobutyrate and pyruvate; acetyl-CoA, propionyl-CoA, butyryl-CoA. malonyl-CoA, valeryl-CoA, and crotonyl-CoA. -Ketoisocaproate, however, is a strong inhibitor of the enzyme. All reactions catalyzed by isopropylmalate synthase were inhibited to the same extent by the endproduct l-leucine. the substrate saturation curves of -ketoisovalerate or other -keto acids and of acetyl-coenzyme A or other acyl-CoA derivatives had intermediary plateau regions; the Hill coefficient alternated between n _H -values higher and lower than 1.0, indicating changes from positive to negative and from negative to positive cooperativity for the substrates. The products, isopropylmalate and free coenzyme A, showed competitive inhibition patterns against both substrates (-ketoisovalerate and acetyl-CoA). Free coenzyme A (1 M) inactivated the enzyme irreversibly. The 3-phosphate of coenzyme A and the free carboxyl group of -ketoisovalerate were involved in optimal binding of these substrates, but 3-dephospho-acetyl-coenzyme A and the methylester of -ketoisovalerate were also converted by this enzyme. A CH₃–CH₂-grouping of the -keto acids seemed to be necessary for binding this substrate.Abbreviations Used CoA Coenzyme A - Tris Tris(hydroxymethyl)aminomethane hydrochloride - DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - IPM -Isopropylmalate - KIV -Ketoisovalerate Prepared from doctoral thesis of the University of Göttingen 1973     

Position-specific interaction between cells of the imaginal wing and haltere discs of Drosophila melanogaster.

When fragments of the imaginal wing disc from opposite ends of the disc are mixed prior to culture, intercalary regeneration occurs so that structures are produced which neither of the fragments would have produced if they had been cultured alone. I report here that fragments of the imaginal wing and haltere disc interact in a position-specific way. Mixing of homologous fragments does not result in regeneration, while mixing of fragments from opposite ends of the discs does. Thus the interaction of wing and haltere disc fragments shows the same positional specificity as the mixing of two wing fragments.     

Differentiation capacities of the dorsal metathoracic (haltere) disc ofDrosophila melanogaster

Summary The mature haltere disc, when implanted into a larva of the same age, undergoes metamorphosis along with the host, and produces a haltere, haltere sclerite, notum III, and pleural process. Implants ordinarily form an inverted vesicle, into which the haltere is frequently everted, usually only partly. Many adventitious bristles may be formed which are not foundin situ. It was found thatsc may reduce, andmwh may increase the number of sensilla.On the basis of results obtained from transplanted disc fragments an organ map of the haltere disc was constructed, which shows the presumptive haltere area to be located in the posterio-central part of the disc, while the anterio-medial part forms the thoracic structures dorsal to the haltere, and the posterio-lateral part the thoracic structures ventral to it. The anlage of the adventitious bristles is probably within the notum area. The haltere anlage proper is concentric; the prospective distal capitellum is located in the center, and is surrounded by the anlagen of the more proximal segments.The organ map of the haltere disc is compared with that of other discs. Organ maps seem to be basically concentric. There is a close homology between the maps of the wing and the haltere disc.     

Regulative Properties of Wing Discs from the Vestigial Mutant of Drosophila melanogaster

The vestigial (vg) mutant of Drosophila melanogaster shows reduced wing size and lacks margin structures from the wing blade. The expressivity is temperature-sensitive, more structures being formed at 29°C than at 25°C. There is cell death in the third instar wing disc which to some extent parallels the fate map locations of the structures absent in the adult.
Vestigial wing discs are unable to regenerate margin structures even when given extra time for growth by culturing them in an adult abdomen before metamorphosis. If the region of cell death is excised from the disc before culture, there is still no regeneration of margin structures, indicating that the dead cells do not physically prevent regulation. Furthermore, by metamorphosing young vg wing discs, it was discovered that cells never acquire competence to make margin during wing disc development. Experiments mixing fragments of vg wing disc with non- vg wing disc fragments of ebony multiple wing hairs (e mwh) genotype showed that the vg cells interacted with the e mwh cells and wing blade was intercalated of both genotypes. However, structures such as wing margin, and alar lobe, usually affected in vg wings, were always made from e mwh cells and not from vg cells. Analysis of mutants which are unable to differentiate particular cell types may help us to understand the mechanism of pattern establishment in developing imaginal discs.     

Chitin biosynthesis in imaginal discs cultured in vitro

Summary We have investigated the stimulation of cuticle production by imaginal discs ofPlodia interpunctella in tissue culture. We turned to biochemical methods to assess the quantitative effects of beta-ecdysone on chitin biosynthesis in wing discs incubated with 0.5 C of C¹⁴-glucosamine for the final 24 h of culture.We demonstrated that imaginal discs ofP. interpunctella respond to increasing concentrations of -ecdysone with increased synthesis. The threshold is between 0.01 and 0.1 g/ml of hormone (2×10^–8 M to 2×10^–7 M). These data represent the first demonstration of quantitative biosynthesis of chitin by a developing tissue in vitro in relation to varying amounts of hormone. Additionally, protein synthesis during the -ecdysone-dependent period was necessary for chitin synthesis. This system thus lends itself to a detailed investigation of the control of chitin biosynthesis.We wish to dedicate this paper to the memory of a fine colleague and friend, Dr. Andrzej Dutkowski     

Absence of red-light enhancement of phototropism in pea seedlings at limiting irradiances of blue light

The effect of different light qualities (blue, green, white, red and far-red) on ethylene production in leaf discs and flower petal discs of Begonia × hiemalis cv. Schwabenland Red was studied. All the light qualities, except far-red, reduced the ACC-conversion to ethylene in leaf discs by about 70% at a photosynthetic photon flux density (PPFD) of 20 mol m^–2s^–1.Blue and green light were less inhibitory than white and red light at lower PPFD. In all treatments far-red light at 0.5 mol m^–2s^–1 of photon flux density (PFD) stimulated the ACC-conversion to ethylene in leaf discs by about 60–90% compared to the dark-incubated control. White and red light strongly inhibited the -naphthalene-acetic acid (NAA) stimulated ethylene synthesis in leaf discs. The results may suggest that the ethylene production is controlled by phytochrome in the leaves but not in the petals. Lack of coaction of any light quality with silver ions on ethylene production in leaf and petal discs was also observed.Abbreviations ACC 1-aminocyclopropane-1-carboxylic acid - EFE ethylene forming enzyme - NAA -naphthalene-acetic acid - PFD photon flux density - PPFD photosynthetic photon flux density - RH relative air humidity - SAM S-adenosylmethionine - STS silver thiosulphate     

Quantitative radioautographic study of intracellular localization of calmodulin antagonist,W-7, in Chinese-hamster ovary cells

Summary The purpose of the present study was to analyse quantitatively the localization of calmodulin antagonist, n-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7) in CHO-Kl cells. The cultured CHO-Kl cells were labelled with 1 (16.7 M), 2 (33.4 M), 5 (83.5 M) and 10 Ci/ml (167 M) tritiated W-7. Some cells were preincubated in 10, 50 and 100 M unlabelled W-7 for 30 min and then labelled with 2 or 5 Ci/ml tritiated W-7 for 1 h. The cells were doubly fixed in glutaraldehyde and osmium-tetroxide solution, and embedded in Epon. For light-microscopic radioautography, 2 m-thick sections were wet mounted with radioautographic emulsion and exposed for 1 month. The radioautograms showed that large numbers of silver grains were mainly localized in the cytoplasm as well as in the nucleus. Quantitative analysis demonstrated that, in both the cytoplasm and nucleus, the number of silver grains was dependent on the concentration of the administered tritiated W-7 and the number was dramatically decreased by the pretreatment of unlabelled W-7. These results show that, in CHO-Kl cells, the W-7 binding sites are saturable. It is concluded that W-7 may get into CHO-Kl cells and be bound to a specific protein that may be calmodulin protein.     

Apoptotic wing degeneration and formation of an altruism-regulating glandular appendage (gemma) in the ponerine ant <Emphasis Type="Italic">Diacamma</Emphasis> sp. from Japan (Hymenoptera,Formicidae, Ponerinae)

We here show an example of morphological novelties, which have evolved from insect wings into the specific structures controlling social behaviour in an ant species. Most ant colonies consist of winged queen(s) and wingless workers. In the queenless ponerine ant Diacamma sp. from Japan, however, all female workers have a pair of small thoracic appendages, called gemmae, which are homologous to the forewings and acts as an organ regulating altruism expression. Most workers, whose gemmae are clipped off by other colony members, become nonreproductive helpers, while only a single individual with complete gemmae becomes functionally reproductive. We examined histologically the development of gemmae, and compared it with that of functional wings in males. Female larvae had well-developed wing discs for both fore- and hindwings. At pupation, however, the wing discs started to evaginate and later degenerate. The hindwing discs completely degenerated, while the degeneration of forewing discs was incomplete, leading to the formation of gemmae. The degeneration process involved apoptotic cell death as confirmed by TUNEL assay. In addition, glandular cells differentiated from the epithelial cells of the forewing buds after completion of pupation. The mechanism of developmental transition from wing to gemma can be regarded as an evolutionary gain of new function, which can be seen in insect appendages and vertebrate limbs.Edited by P. Simpson