Looking at Developmental Neurotoxicity Testing from the Perspective of an Invertebrate Embryo

Abstract

Developmental neurotoxicity (DNT) of chemical compounds disrupts the formation of a normal brain. There is impressive progress in the development of alternative testing methods for DNT potential in chemicals, some of which also incorporate invertebrate animals. This review briefly touches upon studies on the genetically tractable model organisms of Caenorhabditis elegans and Drosophila melanogaster about the action of specific developmental neurotoxicants. The formation of a functional nervous system requires precisely timed axonal pathfinding to the correct cellular targets. To address this complex key event, our lab developed an alternative assay using a serum-free culture of intact locust embryos. The first neural pathways in the leg of embryonic locusts are established by a pair of afferent pioneer neurons which use guidance cues from membrane-bound and diffusible semaphorin proteins. In a systematic approach according to recommendations for alternative testing, the embryo assay quantifies defects in pioneer navigation after exposure to a panel of recognized test compounds for DNT. The outcome indicates a high predictability for test-compound classification. Since the pyramidal neurons of the mammalian cortex also use a semaphorin gradient for neurite guidance, the assay is based on evolutionary conserved cellular mechanisms, supporting its relevance for cortical development.

Keywords: Locusta migratoria; axonal pathfinding; directed cell migration; embryo culture; semaphorin.

Figure 1

DNT assay system for axonal elongation and guidance defects of Ti1 pioneer neurons in embryos of Locusta migratoria. The figure is taken with permission from [38]: (a). Timeline of embryonic development from fertilization (day 0, 0%) to hatching (day 12, 100%) at a temperature of 30 °C. Cultured embryos were incubated in test compounds at the end of day 3 for 18 h (orange tag), followed by two different biochemical assays (blue tag) for viability. Fluorescence image shows a DAPI stained embryo with anterior to the left. (b). Elongation score for quantifying the length of pioneer axons in the limb bud is shown in a scoring scheme ranging from 0 to 100 [37]. Soma positions of the two Ti1 pioneer neuron in the tibia, femur Fe guidepost cell, trochanter Tr guidepost cell and the two coxa Cx1 guidepost cells serve as reference points. (c). Chemical-induced guidance defects are scored, including defasciculations (def) of the two axons, pathfinding errors (pf), and retarded growth (rg).