Zebrafish Ace2N-mNeon expression toolkit for in vivo voltage imaging of neuronal synchrony and cardiac maturation

Significance: Bioelectricity is a fundamental biophysical property of cells that shapes embryogenesis through neuronal signaling and tissue patterning. Yet techniques for cell-resolved, millisecond-scale voltage imaging in early embryos remain limited. Aim: To address this need, we developed a zebrafish expression toolkit for the genetically encoded voltage indicator (GEVI) Ace2N-mNeon, which provides membrane-localized fluorescence from 4 hours post-fertilization (hpf) and supports both broad and promoter-driven, cell-type-specific expression. Approach: This toolkit is built as a small Tol2-based promoter panel, placing Ace2N-mNeon under commonly used zebrafish promoters, and can be delivered by standard Tol2 microinjection workflows. This toolkit spans multiple embryonic tissues; here, we focus on neurons and cardiomyocytes as examples. Results: voltage imaging, we recorded neuronal activity from multiple neuronal types at 1 day post-fertilization (dpf) and observed synchronous activity among motor neurons during primary neurogenesis that appeared reduced or absent during secondary neurogenesis. In parallel, from 1 to 4 dpf, atrial and ventricular voltage signals were detectable before overt chamber delineation; chamber-specific action potential propagation sharpened and accelerated with development, indicating cross-tissue generalizability. Conclusions: voltage imaging during early embryonic development, providing a foundation for linking membrane-voltage dynamics to emergent circuit-level properties.