Inverse Drug Screening of Bioelectric Signaling and Neurotransmitter Roles: Illustrated Using aXenopusTail Regeneration Assay

Abstract

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.

Chemical genetics of regeneration: Contrasting temporal effects of CoCl2 on axolotl tail regeneration

“…Amphibians and fish are well‐suited for studies of tissue regeneration, not only because they regenerate damaged tissues, but also because they are amenable to chemical screening. Chemicals can be readily delivered to aquatic fish, frogs, and salamanders for the purpose of altering developmental, cellular, and molecular mechanisms that regulate tissue regeneration 1‐6 . A variety of chemicals with relatively well‐characterized targets and mechanisms of action have been shown to block regeneration.…”

“…Chemicals can be readily delivered to aquatic fish, frogs, and salamanders for the purpose of altering developmental, cellular, and molecular mechanisms that regulate tissue regeneration. [1][2][3][4][5][6] A variety of chemicals with relatively well-characterized targets and mechanisms of action have been shown to block regeneration. These include chemicals that inhibit primary developmental signaling pathways, including retinoic acid, BMP, TGFα, FGF, Wnt, and HSP90.…”

Aquatic Freshwater Vertebrate Models of Epilepsy Pathology: Past Discoveries and Future Directions for Therapeutic Discovery

“…The transparency of zebrafish embryos and Xenopus tadpoles permits live imaging at the organismal level with a number of tissue-specific transgenic lines available to permit tracking of cellular dynamics in vivo [ 26 , 27 ]. High-throughput screens are possible for both drug discovery and toxicology [ 28 , 29 , 30 , 31 , 32 , 33 , 34 ]. A number of compounds identified in zebrafish screens are in early clinical trials further underscoring the translational potential of conducting drug screens in zebrafish [ 35 ].…”

Endogenous Bioelectrics in Development, Cancer, and Regeneration: Drugs and Bioelectronic Devices as Electroceuticals for Regenerative Medicine

“…Proteomic and physiological profiling, together with drug screens (Sullivan and Levin, 2018), are revealing the molecular sources of relevant ion fluxes. Similarly, the transduction of changes in V mem into second messenger pathways is now understood at the cellular level: voltage-regulated neurotransmitter transporters, calcium channels, KRAS clustering, and butyrate-regulated histone deacetylases convert bioelectric events into transcriptional responses [reviewed in Levin et al (2017)].…”