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.
Overall design: For RNA-Seq, mRNA was isolated from livers at the corresponding stages. For this, livers from 30 larvae / replicate were dissected and collected in the lysis buffer from ReliaPrep™ RNA Miniprep Systems (Promega, Z6110). mRNA was isolated from the lysed tissue by following the manufacture’s instruction. cDNA synthesis, library preparation and Illumina sequencing was performed by Eurofins Genomics Europe Sequencing GmbH. Integrity and quantity of the starting mRNA was determined by appropriate methods, e.g. measurement of volume and quantity, gel electrophoresis and/or fluorimeter measurements. Library preparation incorporated adaptor sequence adapters and indexing compatible for Illumina sequencing technology, using proprietary methods of Eurofins Genomics Europe Sequencing GmbH. The protocol for RNA library preparation was based on NEBNext Ultra II Directional RNA Library Prep Kit for Illumina. For cDNA library preparation, after first strand synthesis, the second strand synthesis was performed using dUPT. The ends of the double stranded cDNA fragments were repaired and dATP ligated to the blunt ended fragments. Next, the sequencing adapters were ligated to the DNA fragments, and the dUTP containing second strand was removed. Sequencing was performed on the Illumina NovaSeq 6000 platform using 2x150 Sequence mode. Raw reads were mapped using HiSAT2 against the GRCz11 zebrafish genome, and counted using FeatureCounts. For normalization, differential gene expression analysis, and GO analysis, iDEP version 0.951 was utilized with default parameters. For differential gene expression, a fold-change of 2 and false-discovery rate of 0.1 was used for cut-off.
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