Rewiring Endogenous Bioelectric Circuits in the Xenopus laevis Embryo Model

Methods Mol Biol. 2021;2258:93-103. doi: 10.1007/978-1-0716-1174-6_7.

Abstract

Embryogenesis, as well as regeneration, is increasingly recognized to be orchestrated by an interplay of transcriptional and bioelectric networks. Spatiotemporal patterns of resting potentials direct the size, shape, and locations of numerous organ primordia during patterning. These bioelectrical properties are established by the function of ion channels and pumps that set voltage potentials of individual cells, and gap junctions (electrical synapses) that enable physiological states to propagate across tissue networks. Functional experiments to probe the roles of bioelectrical states can be carried out by targeting endogenous ion channels during development. Here, we describe protocols, optimized for the highly tractable Xenopus laevis embryo, for molecular genetic targeting of ion channels and connexins based on CRISPR, and monitoring of resting potential states using voltage-sensing fluorescent dye. Similar strategies can be adapted to other model species.

Keywords: Bioelectricity; CRISPR; Frog embryo; Ion channel.

MeSH terms

  • Animals
  • CRISPR-Associated Protein 9 / genetics
  • CRISPR-Associated Protein 9 / metabolism
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • Connexins / genetics
  • Connexins / metabolism*
  • Electrical Synapses / genetics
  • Electrical Synapses / metabolism*
  • Embryo, Nonmammalian / metabolism
  • Embryonic Development
  • Gene Editing*
  • Gene Expression Regulation, Developmental
  • Ion Channels / genetics
  • Ion Channels / metabolism*
  • Membrane Potentials
  • Microscopy, Fluorescence
  • RNA, Guide / genetics
  • RNA, Guide / metabolism
  • Time Factors
  • Xenopus laevis / embryology
  • Xenopus laevis / genetics
  • Xenopus laevis / metabolism*

Substances

  • Connexins
  • Ion Channels
  • RNA, Guide
  • CRISPR-Associated Protein 9