Inverse Drug Screening of Bioelectric Signaling and Neurotransmitter Roles: Illustrated Using a Tail Regeneration Assay.

embryos and larvae are an ideal model system in which to study the interplay between genetics, physiology, and anatomy in the control of structure and function. An important emerging field is the study of bioelectric signaling, the exchange of ion- and neurotransmitter-mediated messages among all types of cells (not just nerve and muscle cells), in the regulation of growth and form during embryogenesis, regeneration, and cancer. To facilitate the mechanistic investigation of bioelectric events in vivo, it is necessary to identify the endogenous signaling machinery involved in any patterning process of interest. This protocol uses the tail regeneration assay in to perform an inverse drug screen; tiers of known compounds are used to probe the involvement of increasingly specific classes of bioelectric and neurotransmitter machinery. By using a hierarchical approach, large classes of targets are ruled out in early rounds, focusing attention on progressively narrower sets of proteins. Such a screen avoids many of the limitations of a molecular-genetic targeting approach and provides a rapid and efficient way to focus on specific targets. Usually, <10 experiments are needed to determine whether bioelectrics and/or neurotransmitter signaling are involved in the process of interest. This protocol describes the strategy in the context of a semiquantitative analysis of tail regeneration but can be applied to any assay in or other small aquatic model system (e.g., zebrafish). Given the ever-increasing toolkit of chemical genetics, such screens represent a powerful and versatile methodology for probing the physiological circuits underlying pattern regulation.