Andreas Dübendorfer

Genetic transformation of the housefly Musca domestica with the lepidopteran derived transposon piggyBac.

The piggyBac transposable element was successfully used for stable genetic transformation of the housefly Musca domestica. The construct contains the EGFP marker under the control of Pax‐6 binding sites, which can drive eye‐specific expression in insect species as distantly related as Drosophila melanogaster and Tribolium castaneum[ Berghammer, A.J., Klingler, M. and Wimmer, E.A. (1999)Nature 402: 370–371]. We obtained seven independent integration events among 41 fertile G0Musca flies. Most of the transformed lines contained two or more chromosomal insertions of the EGFP marker which were stably inherited over more than 15 generations. piggyBac‐mediated transposition was verified by identifying the characteristic TTAA duplication at the insertion sites. This first report of stable transmission of a genetic marker in Musca confirms the use of this vector‐marker system for effective gene transfer in a broad range of insect species.

Gynandromorphs reveal two separate primordia for male and female genitalia inDrosophila melanogaster

SummaryThe derivatives of 110 mosaic genital discs of gynandromorphs have been analysed microscopically. It has been found that theanalia of both sexes are homologous and derive from a single primordium (see Fig. 1a). Whether male or female anal plates are formed depends on the genetic constitution of the cells. This is analogous to the development of male sex combs versus female transversal rows on the forelegs of gynandromorphs. In contrast, the data for thegenitalia (see Fig. 1 b) are best explained if it is assumed that there are two genital primordia in everyDrosophila embryo: a male primordium that will only develop into genitalia if populated by XY (or XO) nuclei, and a female primordium that will only do so if populated by XX nuclei. This model, as depicted in Figure 2, is compatible with all our gynandromorph data and also with observations onMusca andCalliphora where in fact two separate genital primordia are found.

Ecdysteroids in adult males and females of Drosophila melanogaster

Ecdysteroid titres in whole flies and different tissues of adult male and female Drosophila were determined at various times after eclosion using a radioimmunoassay. The ecdysteroid titre decreased as the flies matured after eclosion. The differences in titre between males and females can be accounted for by their difference in body weight. The ecdysteroids were found to be distributed throughout several tissues. At eclosion not all of the ecdysteroid complement present could be accounted for by that found localised in tissues. After maturation of the flies the ecdysteroids in various tissues can account for the majority of that detected in whole-fly extracts. Ecdysteroids were produced during in vitro culture of various tissues, but the quantities detected were low by comparison with ring glands of wandering 3rd-instar larvae. Neither the ovaries nor the abdominal body walls (fat body) seem to be a major source of hormone, and they are only able to convert minute quantities of ecdysone to the biologically active form, 20-hydroxyecdysone, in vitro. The amounts of 20-hydroxyecdysone present were measured using high performance liquid chromatography and radioimmunoassay. We tentatively suggest that the differential experession of the yolk-protein-genes in the fat bodies of males and females does not result from differences in hormone titres between them.

The transformer2 gene in Musca domestica is required for selecting and maintaining the female pathway of development

We present the isolation and functional analysis of a transformer2 homologue Mdtra2 in the housefly Musca domestica. Compromising the activity of this gene by injecting dsRNA into embryos causes complete sex reversal of genotypically female individuals into fertile males, revealing an essential function of Mdtra2 in female development of the housefly. Mdtra2 is required for female-specific splicing of Musca doublesex (Mddsx) which structurally and functionally corresponds to Drosophila dsx, the bottom-most regulator in the sex-determining pathway. Since Mdtra2 is expressed in males and females, we propose that Mdtra2 serves as an essential co-factor of F, the key sex-determining switch upstream of Mddsx. We also provide evidence that Mdtra2 acts upstream as a positive regulator of F supporting genetic data which suggest that F relies on an autocatalytic activity to select and maintain the female path of development. We further show that repression of male courtship behavior by F requires Mdtra2. This function of F and Mdtra2 appears not to be mediated by Mddsx, suggesting that bifurcation of the pathway at this level is a conserved feature in the genetic architecture of Musca and Drosophila.

Embryonic ecdysteroids of Drosophila melanogaster

Abstract Titration by RIA of ecdysteroids during embryogenesis of Drosophila shows one single peak at 8 h, i.e. the time of germ-band shortening. An analysis of the ecdysteroids reveals a general shift from low polarity material at 6 h to free ecdysone and (less) 20-hydroxyecdysone at 8 h, and finally to ecdysteroids of mostly higher polarity at 12 h. Adult body walls, when incubated with tritiated 20-hydroxyecdysone, can produce the 22-acetate of this ecdysteroid which then has similar chromatographic properties as ecdysone. We have isolated the putative ecdysone fraction from 8 h embryonic extracts and showed by enzymatic hydrolysis and HPLC that the embryo does not contain 20-hydroxyecdysone-22-acetate, but rather free ecdysone.

Differentiation in vitro of larval and adult muscles from embryonic cells ofDrosophila

SummaryThe differentiation of muscles in primary cultures of cells fromDrosophila melanogaster embryos was investigated. In early cultures, and in the absence of exogenous ecdysone, two main classes of muscle were found. Comparison, by light and electron microscopy, of one of these classes (the “myotube” class) with muscles from third instar larvae shows that this class corresponds to the muscles of the body wall of the larva. When α- or β-ecdysone is added to the cultures, these undergo a number of metamorphic changes. Most of the larval muscles disappear, and two new types of muscle form. Ultrastructural and light microscopic examination of these two types indicates that they correspond to the two classes of skeletal muscle (fibrillar and tubular) found in adult flies.

Ecdysteroid conjugation by tissues of adult females of Drosophila melanogaster

Abstract In order to determine whether female adults of Drosophila can synthesize enzymatically cleavable ecdysteroid conjugates, we injected radiolabelled ecdysone into flies and cultured various organs in vitro in the presence of labelled ecdysteroids. Within 1 hr most of the injected ecdysone was metabolized to 20-hydroxyecdysone, high polarity products (HPP), low polarity products (LPP) and some highly apolar compounds (AP). Incubation of organs from axenically reared flies (abdominal carcass, gut, Malpighian tubules, ovaries) in a culture medium containing tritiated ecdysteroids revealed tissue-specific conjugate synthesis. HPP was found in all tissues, but was by far the most prominent metabolite in ovary cultures. All tissues except the ovaries also contained labelled LPP which appeared bound to the cells, especially of the carcass and the gut. HPP and LPP were synthesized from both ecdysone and 20-hydroxyecdysone in vitro . They were shown to be ecdysteroid conjugates by enzymatic hydrolysis: HPP was cleaved by Helix pomatia hydrolases, and LPP was digestible with porcine liver esterase. Whether any of these conjugates have specific functions in supplying the eggs with ecdysteroid precursors is not yet known.

Development of imaginal discs from lethal hybrids betweenDrosophila melanogaster andDrosophila mauritiana

SummaryFemales ofDrosophila melanogaster, crossed with males ofDrosophila mauritiana, produce only female offspring. The male hybrid larvae grow very slowly, fail to pupate and die after prolonged larval life. Imaginal discs from these male hybrids transplanted into Drosophila melanogaster larvae can give rise to adult structures with normal patterns. Differentiation of hybrid imaginal disc tissue is improved by short term culture in non-hybrid larvae prior to metamorphosis, suggesting that the hybrid larval haemolymph is inadequate to sustain normal imaginal disc growth. This may represent the physiological basis of the reproductive isolating mechanism separating the twoDrosophila species

Cell differentiation in vitro and establishment of permanent, ecdysone-responsive cell lines from embryonic tissues of the Colorado potato beetle, Leptinotarsa decemlineata

Abstract When embryos of the shortened germ-band stage of the Colorado potato beetle, Leptinotarsa decemlineata , were mechanically broken up into small fragments and placed in Drosophila tissue culture medium, cells migrated out from the fragments and populated the culture chamber. Many cells differentiated into larval neurones, myocytes, haemocytes and epidermal cells. Subsequently, tissues such as ganglia, muscles, tracheae and larval epidermis with fully differentiated larval bristles developed without exogenous ecdysteroid hormones being applied. Perfect patterning of a cuticular structure (labium) resulted once in a culture that was treated with ecdysone. In the second week after setting up, many cultures produced masses of cellular vesicles which were maintained in vitro for several months. When the vesicles collapsed, undifferentiated, polygonal and spindle-shaped cells grew out and multiplied to confluency. From this material, we have established a permanent line (ZIZ-LD-1) with three sublines (1–3, 1–4 and 1–15) of L. decemlineata cells with a doubling time of 5 days. Upon treatment with ecdysone, the cells of the permanent line ZIZ-LD-1-4 aggregated, whilst control cultures remained disperse. This reaction must have been elicited by ecdysone itself since the cells did not hydroxylate ecdysone into 20-hydroxyecdysone, even after incubation periods of up to 5 days.

Distribution of heterochromatin on the mitotic chromosomes of Musca domestica L. in relation to the activity of male-determining factors

Abstract. In the housefly, male sex is determined by a dominant factor, M, located either on the Y, on the X, or on any of the five autosomes. M factors on autosome I and on fragments of the Y chromosome show incomplete expressivity, whereas M factors on the other autosomes are fully expressive. To test whether these differences might be caused by heterochromatin-dependent position effects, we studied the distribution of heterochromatin on the mitotic chromosomes by C-banding and by fluorescence in situ hybridization of DNA fragments amplified from microdissected mitotic chromosomes. Our results show a correlation between the chromosomal position of M and the strength of its male-determining activity: weakly masculinizing M factors are exclusively located on chromosomes with extensive heterochromatic regions, i.e., on autosome I and on the Y chromosome. The Y is known to contain at least two copies of the M factor, which ensures a strong masculinizing effect despite the heterochromatic environment. The heterochromatic regions of the sex chromosomes consist of repetitive sequences that are unique to the X and the Y, whereas their euchromatic parts contain sequences that are ubiquitously found in the euchromatin of all chromosomes of the complement.