Collin Murphy

The synthetic and minimal culture requirements for evagination of imaginal discs of Drosophila melanogaster in vitro

Abstract Imaginal discs are induced by β-ecdysone to evaginate and undergo imaginal differentiation in completely defined culture medium (Robbs). The minimal nutritional requirements for evagination are salts, glucose, and 6 or 7 amino acids. Concentrations of β-ecdysone which cause evagination also produce increases in RNA and protein synthesis. Inhibitors of RNA and protein synthesis and amino acid starvation block evagination. Inhibitors of DNA synthesis do not inhibit evagination. The effects of β-ecdysone are concentration dependent. To produce complete evagination, discs must be exposed to low concentrations (0.1 μg/ml) of β-ecdysone for a longer time than to high concentrations (10 μg/ml). However, high concentrations of hormone reduce the rate, and under some conditions, the degree of evagination.

The recovery and preliminary characterization of X chromosome mutants affecting imaginal discs ofDrosophila melanogaster

A selection method especially designed for isolation of X-linked lethals in Drosophila having defective imaginal discs has generated 26 mutants with high larval viability, but which terminate development in late larval and prepupal stages. The mutants are tentatively classified into four categories, according to the morphological appearance of their imaginal discs in third-instar larvae, and are further characterized with reference to autonomy/nonautonomy and to capacity for in vitro evagination of discs. The four categories are as follows: 1. discs degenerate = extreme reduction of disc tissue (8 mutants). Of 7 mutants tested, all appear to be autonomous. 2. discs small = reduction in size of disc tissue (7 mutants). Of 6 mutants tested, 5 are nonautonomous. However, discs from 4 of the nonautonomous mutants fail to evaginate in vitro . 3. discs large = hypertrophy of disc tissue (1 mutant). The mutant is phenotypically similar to an allele of lethal giant larvae (l(2)gl 4 ) . 4. discs normal = normal appearance of disc tissue (10 mutants). Of 9 mutants tested, 5 are nonautonomous. Discs from one mutant evaginate abnormally in vitro and in vivo .

The ultrastructure of the differentiating pupal leg ofDrosophila melanogaster

SummaryThe ultrastructure of the developing pupal leg ofDrosophila melanogaster is investigated at various stages from white prepupa to pharate adult. All the different cell types found in the leg at each stage examined are traced developmentally. This includes general epithelium which secretes both a pupal and adult cuticle and specialized epithelial structures such as apodemes, bristles and other sense organs. Muscle, nerve and tracheal tissue develop within the leg cavity. Particular attention was paid to the development of muscle tissue which was shown to differentiate from the adepithelial cells of the larval disc. The development of theDrosophila leg is compared with that ofCalliphora (van Ruiten and Sprey, 1974) especially with respect to the different stages at which new structures appear in the two species.

Cell death and autonomous gene action in lethals affecting imaginal discs in Drosophila melanogaster

A series of 23 X-linked lethals affecting imaginal discs, previously classified as to disc phenotype and autonomy/nonautonomy of lethal gene action has been histologically characterized. Fluorescence microscopy of whole mounts of discs from 22 of the mutants and examination of sectioned disc material from 15 of the mutants at late third instar reveals the following: Thirteen of the mutants have extensive cell death; 10 of these mutants act autonomously (cell lethality). Three of the mutants with cell death phenotypes were previously classified as “normal” as judged by dissection. Discs from these three mutants may be able to respond to the molting hormone, β-ecdysone, but may be deficient in a secondary response leading to death of the tissue. No histologically normal autonomous mutants were found. Ten mutants have normal patterns of degeneration and function nonautonomously. These mutants may have defects external to the disc tissue. The autonomous mutants tend to have a slightly earlier effective lethal phase than the nonautonomous mutants.

Determination of the dorsal mesothoracic disc inDrosophila

Extirpations of the dorsal mesothoracic disc carried out by Pantelouris and Waddington in mature larvae ofDrosophila melanogaster resulted in the formation of mesonotal chaetae on the operated side in 4 out of 20 cases. This experiment was repeated on a large scale in both mature and 72-hour third instar larvae. Special attention was paid to recording the degree of disc removal in these operations. Among the 137 survivors of the extirpations, 126 (92%) developed no more than one wing and one-half of the mesonotum. The 11 cases in which mesonotal chaetae or wing structures developed on the operated side all (except an unclassified specimen) belonged to classes in which partial extirpation of the disc had been scored. The structures which differentiated on the extirpated side generally conformed to the specific presumptive parts within the disc anlage which remained inside the larval body in the various extirpation classes. Both observations indicate that the structures which developed on the operated side arose from remnants of the partially extirpated disc. In contrast to the suggestion of Pantelouris and Waddington, evidence has been presented which indicates that the dorsal mesothoracic discs do not exist as members of a bicentric regulative field. Instead, it is concluded that determination of the pair of wing discs as right and left entities is complete, not only in mature larvae, but also in larvae in the middle of the third instar. Similar conclusions are drawn with respect to the paired eye discs. Among specimens in which chaetae did not develop on the operated side, regulation limited to cell proliferation and epidermal spreading may have occurred in 2 individuals in the Pantelouris and Waddington experiment and in 9 cases in the present study. This growth might be interpreted as a homeostatic response of trichome anlagen to absence of cellular material from the operated disc at the time of normal fusion of mesothoracic disc products.

Aspects of development of imaginal discs in a non-pupariating lethal mutant in Drosophila melanogaster

Abstract Imaginal discs from a non-pupariating mutant, lethal-1 a , were cultured in vitro. Responses of the discs to B-ecdysone are abnormal, including failure to maintain an increased level of RNA synthesis, depression of protein synthesis and cell death. When lethal-1 a discs are transplanted into wild-type larvae for metamorphosis, almost all bristle-forming regions fail to differentiate, whereas non-bristle-forming regions do so normally. Mutant larvae apparently secrete molting hormone, and can synthesize cuticle within unevaginated discs. The in vitro experiments suggest that abnormal responses of mutant tissue to β-ecdysone in situ result in non-pupariation, failure to disc evagination and death.