LiverZap: a chemoptogenetic tool for global and locally restricted hepatocyte ablation to study cellular behaviours in liver regeneration.

The liver restores its mass and architecture after injury. Yet, investigating morphogenetic cell behaviours and signals that repair tissue architecture at high spatiotemporal resolution remains challenging. We developed LiverZap, a tuneable chemoptogenetic liver injury model in zebrafish. LiverZap employs the formation of a binary FAP-TAP photosensitiser followed by brief near-infrared illumination inducing hepatocyte-specific death and recapitulating mammalian liver injury types. The tool enables local hepatocyte ablation and extended live imaging capturing regenerative cell behaviours, which is crucial for studying cellular interactions at the interface of healthy and damaged tissue. Applying LiverZap, we show that targeted hepatocyte ablation in a small region of interest is sufficient to trigger local liver progenitor-like cell (LPC)-mediated regeneration, challenging the current understanding of liver regeneration. Surprisingly, the LPC response is also elicited in adjacent uninjured tissue, at up to 100 µm distance to the injury. Moreover, dynamic biliary network rearrangement suggests active cell movements from uninjured tissue in response to substantial hepatocyte loss as an integral step of LPC-mediated liver regeneration. This precisely targetable liver cell ablation tool will enable the discovery of key molecular and morphogenetic regeneration paradigms.

Lineage tracing identifies heterogeneous hepatoblast contribution to cell lineages and postembryonic organ growth dynamics.

To meet the physiological demands of the body, organs need to establish a functional tissue architecture and adequate size as the embryo develops to adulthood. In the liver, uni- and bipotent progenitor differentiation into hepatocytes and biliary epithelial cells (BECs), and their relative proportions, comprise the functional architecture. Yet, the contribution of individual liver progenitors at the organ level to both fates, and their specific proportion, is unresolved. Combining mathematical modelling with organ-wide, multispectral FRaeppli-NLS lineage tracing in zebrafish, we demonstrate that a precise BEC-to-hepatocyte ratio is established (i) fast, (ii) solely by heterogeneous lineage decisions from uni- and bipotent progenitors, and (iii) independent of subsequent cell type-specific proliferation. Extending lineage tracing to adulthood determined that embryonic cells undergo spatially heterogeneous three-dimensional growth associated with distinct environments. Strikingly, giant clusters comprising almost half a ventral lobe suggest lobe-specific dominant-like growth behaviours. We show substantial hepatocyte polyploidy in juveniles representing another hallmark of postembryonic liver growth. Our findings uncover heterogeneous progenitor contributions to tissue architecture-defining cell type proportions and postembryonic organ growth as key mechanisms forming the adult liver.

FRaeppli: a multispectral imaging toolbox for cell tracing and dense tissue analysis in zebrafish.

Visualizing cell shapes and interactions of differentiating cells is instrumental for understanding organ development and repair. Across species, strategies for stochastic multicolour labelling have greatly facilitated in vivo cell tracking and mapping neuronal connectivity. Yet integrating multi-fluorophore information into the context of developing zebrafish tissues is challenging given their cytoplasmic localization and spectral incompatibility with common fluorescent markers. Inspired by Drosophila Raeppli, we developed FRaeppli (Fish-Raeppli) by expressing bright membrane- or nuclear-targeted fluorescent proteins for efficient cell shape analysis and tracking. High spatiotemporal activation flexibility is provided by the Gal4/UAS system together with Cre/lox and/or PhiC31 integrase. The distinct spectra of the FRaeppli fluorescent proteins allow simultaneous imaging with GFP and infrared subcellular reporters or tissue landmarks. We demonstrate the suitability of FRaeppli for live imaging of complex internal organs, such as the liver, and have tailored hyperspectral protocols for time-efficient acquisition. Combining FRaeppli with polarity markers revealed previously unknown canalicular topologies between differentiating hepatocytes, reminiscent of the mammalian liver, suggesting common developmental mechanisms. The multispectral FRaeppli toolbox thus enables the comprehensive analysis of intricate cellular morphologies, topologies and lineages at single-cell resolution in zebrafish.

Kupffer Cells Sense Free Fatty Acids and Regulate Hepatic Lipid Metabolism in High-Fat Diet and Inflammation.

A high fat Western-style diet leads to hepatic steatosis that can progress to steatohepatitis and ultimately cirrhosis or liver cancer. The mechanism that leads to the development of steatosis upon nutritional overload is complex and only partially understood. Using click chemistry-based metabolic tracing and microscopy, we study the interaction between Kupffer cells and hepatocytes ex vivo. In the early phase of steatosis, hepatocytes alone do not display significant deviations in fatty acid metabolism. However, in co-cultures or supernatant transfer experiments, we show that tumor necrosis factor (TNF) secretion by Kupffer cells is necessary and sufficient to induce steatosis in hepatocytes, independent of the challenge of hepatocytes with elevated fatty acid levels. We further show that free fatty acid (FFA) or lipopolysaccharide are both able to trigger release of TNF from Kupffer cells. We conclude that Kupffer cells act as the primary sensor for both FFA overload and bacterial lipopolysaccharide, integrate these signals and transmit the information to the hepatocyte via TNF secretion. Hepatocytes react by alteration in lipid metabolism prominently leading to the accumulation of triacylglycerols (TAGs) in lipid droplets, a hallmark of steatosis.

Intron with transgenic marker (InTraM) facilitates high-throughput screening of endogenous gene reporter lines.

The generation and maintenance of genome edited zebrafish lines is typically labor intensive due to the lack of an easy visual read-out for the modification. To facilitate this process, we have developed a novel method that relies on the inclusion of an artificial intron with a transgenic marker (InTraM) within the knock-in sequence of interest, which upon splicing produces a transcript with a precise and seamless modification. We have demonstrated this technology by replacing the stop codon of the zebrafish fli1a gene with a transcriptional activator KALTA4, using an InTraM that enables red fluorescent protein expression in the heart.