In an effort to optimize our assays of MeHg toxicity we have innovated a method for routine pharmacological application of small molecules (i.e. drugs and toxins) to the developing Drosophila embryo, a premier model of developmental biology. We have exploited this system for both phenotype characterization and for gene discovery in the context of MeHg toxic exposures. Our initial studies using embryos revealed signaling molecules downstream of the Notch receptor pathway, the enhancer of split (E(spl)) genes, as targets of MeHg exposure in the embryo.
Pictured Right: Determination of permeabilization and methylmercury effects in fixed and immunostained embryos. (see Rand, JoVE, 2014, in press) Embryos were collected at 25°C for 2 hours and aged for 14 hours at 18°C (equivalent to 7-9 hour embryos at 25°C, stage 12). Permeabilization treatment was done followed by incubation in CY5 dye (50µM for 15 minutes) as a permeability indicator together with methylmercury (50µM MeHg, Panel B) or DMSO solvent control (Panel A). Embryos were developed for an additional 8 hours at room temperature. Embryos were then fixed and stained with anti-Fasciclin II (green in A,B and white in A’,B’) to label motor neurons and anti-elav antibodies (red in A, B) to label all neuron cell bodies. CY5 dye is revealed by direct fluorescence, which requires extended exposure due to diminished fluorescence intensity due to fixation (CY5 is pseudo-colored blue in all panels). The effects of MeHg are seen in the irregular patterning and clustering of the lateral chordotonal neuron cell bodies (elav-positive, labeled in red and denoted with white arrows in B versus A). In addition, a characteristic branching of the segmental (SN) (solid green arrows in A’) is seen to be highly variable with MeHg exposure (solid green arrows in B”) consistent with previously reported effects of MeHg on the embryo (see Engel, Tox In Vitro, 2012). Projection of the intersegmental and segmental nerves at their roots are seen to be displaced posteriorly with MeHg exposure (open green arrow in B’). (Embryo orientation: anterior is right, dorsal is up)
We have recently characterized one of these genes, E(spl)mδ, which shows muscle-specific expression and has influence over neuromuscular development. Ongoing studies using this approach are focused on pinpointing distinct windows of MeHg susceptibility during embryogenesis. This methodology also offers an exciting avenue of assay development and the potential for a high-throughput, high-information content platform with wider implications for advancing toxicology and drug studies in multiple arenas.