Bioelectricity in Developmental Patterning and Size Control: Evidence and Genetically Encoded Tools in the Zebrafish Model

Abstract

Developmental patterning is essential for regulating cellular events such as axial patterning, segmentation, tissue formation, and organ size determination during embryogenesis. Understanding the patterning mechanisms remains a central challenge and fundamental interest in developmental biology. Ion-channel-regulated bioelectric signals have emerged as a player of the patterning mechanism, which may interact with morphogens. Evidence from multiple model organisms reveals the roles of bioelectricity in embryonic development, regeneration, and cancers. The Zebrafish model is the second most used vertebrate model, next to the mouse model. The zebrafish model has great potential for elucidating the functions of bioelectricity due to many advantages such as external development, transparent early embryogenesis, and tractable genetics. Here, we review genetic evidence from zebrafish mutants with fin-size and pigment changes related to ion channels and bioelectricity. In addition, we review the cell membrane voltage reporting and chemogenetic tools that have already been used or have great potential to be implemented in zebrafish models. Finally, new perspectives and opportunities for bioelectricity research with zebrafish are discussed.

Keywords: GEVI; bioelectricity; chemogenetics; embryonic development; ion channels; long fin; optogenetics; patterning; pigment; short fin; zebrafish.

Figure 1

Cell membrane potential formation and comparison of neuromuscular excitable cells and non-excitable somatic cells. (A). Illustration of resting membrane potential, ion regulators, and ionic concentrations when the cell is in a non-excitable state. Different shapes represent various ion regulators on a cell membrane (blue region). The arrows indicate the movement of ions when the regulators are open. (B). Comparison of neuromuscular excitable and non-excitable somatic cells. Excitable cells usually exhibit action potentials, while the non-excitable somatic cells have membrane potential fluctuations, which vary in their amplitudes and frequencies.

Figure 2

Illustrations of design principles of common GEVIs, optogenetic and chemogenetic tools. (A). Schematic structures of VSD (voltage sensitive domain) based and opsin-based GEVIs. The voltage-sensitive domain is labeled brown in the cell membrane. A fluorescent protein (FP) is inserted into the S3–S4 loop. When Vm is altered, the fluorescence intensity will change accordingly. The light-sensitive opsin (dark blue) can sense the Vm of the membrane and act as a chromophore. Thus, it can be used to measure the Vm with or without a connected FP, which can enhance the overall signal. (B). Principles of Optogenetics and chemogenetics. The optogenetic tools are based on light-sensitive channel rhodopsins that conduct protons or chloride. The PSAMs are mutated ligand-gated ion channels for sodium or chloride. They are controlled by artificial PSEM (pharmacologically selective effector molecules) ligands. In contrast, the DREADDs are mutated ligand-gated GPCRs (G-protein-coupled receptors). Depending on the type of G protein, they can increase or decrease Vm via GIRK channels, calcium signaling, and cAMPs. The arrows indicate the movement of ions when the regulators are open.

Figure 3

Summary and perspectives of bioelectricity in zebrafish developmental patterning research. Bioelectricity may function in zebrafish early embryos for patterning adult tissue/organs. Multiple ion regulators exist in each cell, so the cell’s bioelectric properties are generally robust. Minor disruptions (smaller lightning arrows) are not enough to change the bioelectric status quo, and only significant changes (big lightning arrows) may break through the robustness. This may explain why most zebrafish mutants were found with GOF (gain-of-function), DN (dominant negative), and ectopic expressions. Mechanistically, this bioelectric patterning may interact with already-known morphogen proteins and transcription factors. However, most likely, there are unknown mechanisms that mediate this bioelectric patterning. Next-generation sequencing technologies and CRISPR genome editing may help decipher such novel mechanisms. In addition, the recently developed genetically encoded tools for neuroscience, such as GEVIs, optogenetics, and chemogenetics, are readily adopted to the embryonic patterning research field. These tools allow us to monitor and manipulate bioelectricity in a non-invasive manner.