In vivo imaging of calcium dynamics in zebrafish hepatocytes

Abstract

Hepatocytes were the first cell-type for which oscillations of cytoplasmic calcium levels in response to hormones were described. Since then, investigation of calcium dynamics in liver explants and culture has greatly increased our understanding of calcium signaling. A bottleneck, however, exists in observing calcium dynamics in a non-invasive manner due to the optical inaccessibility of the mammalian liver. Here we take advantage of the transparency of the zebrafish larvae to develop a setup that allows in vivo imaging of calcium flux in zebrafish hepatocytes at cellular resolution. Using this, we provide quantitative assessment of intracellular calcium dynamics during multiple contexts, including growth, feeding, ethanol-induced stress and cell ablation. Specifically, we show that synchronized calcium oscillations are present in vivo, which are lost upon starvation. Feeding recommences calcium waves in the liver, but in a spatially restricted manner. Further, ethanol treatment as well as cell ablation induces calcium flux, but with different dynamics. The former causes asynchronous calcium oscillations, while the latter leads to a single calcium spike. Overall, we demonstrate the presence of oscillations, waves and spikes in vivo. Thus, our study introduces a platform for observing diverse calcium dynamics while maintaining the native environment of the liver, which will help investigations into the dissection of molecular mechanisms supporting the intra-and intercellular calcium signaling in the liver.

Starvation resistant cavefish reveal conserved mechanisms of starvation-induced hepatic lipotoxicity

“…We found that at 4 dpf, zebrafish display very little hepatic lipid droplets, while at 6 dpf, without exogenous feeding, zebrafish enter a fasting state with robust hepatic steatosis (Fig. 2A) (13). Following 6 dpf, hepatic lipid droplets decreased (Fig.…”

“…However, to our knowledge, it is not known if liver damage upon starvation progresses through a similar sequence of events. To investigate this, we turned to the zebrafish as a model system, where we and others have shown robust development of starvation-induced hepatic steatosis (13,14), similar to surface fish. We visualized hepatic lipid droplets in the Tg(fabp10a:EGFP) reporter line, which marks hepatocytes with green fluorescence, using Nile Red staining of neutral lipids.…”

The enhanced hepatotoxicity of isobavachalcone in depigmented zebrafish due to calcium signaling dysregulation and lipid metabolism disorder

“…Maus et al (2017) observed that calcium homeostasis disruption led to the accumulation of lipid droplets in the liver, heart, and skeletal muscles of mice. Pozo-Morales et al (2023) found that the inhibition of calcium signaling accelerated the accumulation of starvation-related lipid droplets in the zebrafish liver through a genetically encoded calcium scavenger. Adequate mitochondrial function could maintain cardiovascular health and normal metabolism (Lane, 2006), as mitochondria constantly absorbed Ca 2+ to meet their physiological needs (Prudent et al, 2015).…”

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

“…Calcium has also been widely used to reflect neuronal electric activities. Genetically encoded calcium indicators, GECIs, have been widely implemented into the zebrafish model for neural studies [ 147 , 148 , 149 ], cell migration [ 150 , 151 ], embryogenesis [ 152 , 153 ], insulin secretion from pancreatic beta cells [ 154 ], and many other biological processes [ 155 , 156 , 157 , 158 ]. Although calcium signals mimic electric signals, they are still different due to their additional function as a secondary messenger in various cell types.…”