CONDITIONS FOR THE PRIMARY CULTURE OF EYE IMAGINAL DISCS FROM DROSOPHILA-MELANOGASTER

We have established a primary culture system for Drosophila eye imagin al discs, With this system, we were able to obtain neurite outgrowth f rom intact eye discs, eye disc fragments, and dissociated eye imaginal disc cells, Immunoreactivity to antibody 24B10 indicates that these e xtending neurites are photoreceptor axons. Three culture media were te sted for their ability to support the survival of and neurite extensio n from eye disc fragments in vitro at 23 degrees C. These, with supple ments, were: five parts of Schneider's Drosophila medium with four par ts of basal Eagle's medium (''4+5''); Leibovitz's L-15 medium (L-15); and Shields and Sang's M3 modified medium (MM3). We obtained the best results with MM3 supplemented with 2% fetal bovine serum (FBS), Eye di sc fragments survived in this medium for at least 20 days. Pigmentatio n in the nonphotoreceptor pigment cells in cultures from the prepupa r equired the presence of 20-hydroxyecdysone (20-HE) (1 mu g/ml), wherea s neurite outgrowth was seen in the absence of 20-HE. Donor animals ha d to fall within a range of ages to obtain appropriate eye disc differ entiation in vitro. Eye discs from 5-h pupae (P+5) or older commenced omma-chrome synthesis in vitro in a temporal sequence close to that fo und in vivo, whereas the in vitro synthesis of this pigment was delaye d in eye discs from younger flies. Average neurite length was not affe cted by age among pupae younger than P+5; but neurite outgrowth from P +24 was scarce, probably because by this time photoreceptor axons had already grown in vivo and were severed and unable to regenerate in vit ro, Eye discs taken from third instar larvae or white prepupae continu ed their mitotic activity in vitro, Together with the advance of the m orphogenetic furrow at the leading edge of retinal development, this o bservation is consistent with the evidence that pattern formation cont inues in vitro, Morphogenetic changes were manifested in cultures, Via bility tests with calcein AM and ethidium bromide revealed few dead ce lls in living cultures. (C) 1995 John Wiley & Sons, Inc.