164 Dorsoventral asymmetry in mitochondrial membrane potential in early Drosophila embryos

Abstract

The correct establishment of dorsoventral (DV) polarity in the early Drosophila embryo has. through extensive genetic and molecular studies, been found to depend on a set of I 1 'dorsal-group' genes and the gene, cacrus [ I ] ; the products of these genes together form a signal transduction pathway that results in a ventrodorsal nuclear gradient of Dorsal, a transcription factor of the RellNFkt? family. This signalling pathway shares many features with that of the interleukin-l -mediated activation of NFKB. The resulting nuclear gradient of Dorsal ensures the correct pattern of expression of a group of genes along the dorsal-ventral axis, which is necessary for development to proceed as normal. The work presented here shows the existence of a different kind of gradient, not of proteins but of mitochondrial membrane potential (MMP), as measured by a f l u o r e s c e n t p robe , J C I (5.5'.6,6'-letrachloro-l , l ' , 3 , 3 ' tetraethylbenzimidazolylcarbocyanine iodide) [2.3]. This gradient may represent one of the many signals that act to set up the DV axis in Drosophila. Embryos were stained with JCI using a permeabilisatiodstaining procedure based closely on that discussed in (41. as used in [5], and on advice from K. Whittaker. Drosuphila embryos in a range of developmental stages were collected by placing apple-juice/agar plates for several hours in a population cage; embryos were dechorionated in 50% bleach (J Sainsbury's Ltd.) for about 90 seconds; following a thorough rinse in distilled water. they were left on the filter for several minutes at room temperature. Staining was performed on siliconised cavity slides: onto each was placed 25p1 of staining solution, containing 10pg/ml JC-I and 10% DMSO, in double-distilled water (DDW)). and in some cases 100pg/ml AMC (N-CBZ-4-amino-7-methyl coumarin hydrochlonde). A small amount of n-octane (to permeabilise the embryos) was added to the cavity and a small number of embryos was transferred from the Nitex filter to the octane; most of the octane was then removed using a drawnout pipette. and the slide gently tapped to help align the embryos around the staining solution as much as possible; after the remaining octane was removed or had evaporated. an additional 75p1 of staining solution was added, and the slide left for 10-20 minutes in a humid chamber (a Petri dish containing moist