Periodic Reporting for period 1 - LIVERZAP (Live, In vivo Visualisation of liver Regeneration in Zebrafish After Photoablation of hepatocytes)
Okres sprawozdawczy: 2018-01-01 do 2019-12-31
The liver is an essential metabolic organ with innate regenerative capacity. It has a complex tissue architecture that is crucially important for its function. In higher vertebrates, chronic liver disease and toxic insult leads to gradual compromise of the liver architecture through scarring and cirrhosis for which the only course of treatment is organ transplant. Development of novel therapies requires a detailed understanding of how the liver regenerates after injury in vivo. Prior to this project, studies using animal models of liver regeneration had revealed which hepatic cell types respond to different modes of damage (1-4). However, the cell behaviours and molecular signalling responsible for the restoration of the liver architecture following injury remained largely unclear and were yet to be studied in vivo. The LIVERZAP project pioneered real-time, in vivo image analysis and transcriptomics in zebrafish to examine and quantify the immediate cell behavioural response to liver injury. The fellow built on previous experience in microscopy to establish a deep tissue, multi-colour live imaging pipeline using light sheet microscopy. Cell-type specific molecular profiling provided by RNA-sequencing was employed to identify transcriptionally independent factors controlling the immediate morphogenetic response to liver injury. Together, the strategies employed by LIVERZAP have greatly enhanced the competencies of the fellow in specialized techniques, including state-of-the-art microscopy systems and methodologies, whilst providing excellent new tools for regeneration research. Fundamentally, the outcomes of LIVERZAP have begun to afford essential cellular and mechanistic insights into the restoration of liver architecture following injury, providing a platform for the long-term development of therapies for human liver pathologies.
(1) Yanger, K. et al.(2014) “Adult Hepatocytes Are Generated by Self-Duplication Rather than Stem Cell Differentiation.” Cell Stem Cell,15: 340–349.
(2) Miyaoka, Y. et al. (2012) “Hypertrophy and Unconventional Cell Division of Hepatocytes Underlie Liver Regeneration.” Current Biology,22: 1166–1175.
(3) Español Suñer, R. et al. (2012) “Liver Progenitor Cells Yield Functional Hepatocytes in Response to Chronic Liver Injury in Mice.” Gastroenterology,143: 1564–1575.
(4) Zheng, D. et al. (2006) “Oval Cell Response in 2-Acetylaminofluorene/Partial Hepatectomy Rat Is Attenuated by Short Interfering RNA Targeted to Stromal Cell-Derived Factor-1.” The American Journal of Pathology,169: 2066–2074
(1) Yanger, K. et al.(2014) “Adult Hepatocytes Are Generated by Self-Duplication Rather than Stem Cell Differentiation.” Cell Stem Cell,15: 340–349.
(2) Miyaoka, Y. et al. (2012) “Hypertrophy and Unconventional Cell Division of Hepatocytes Underlie Liver Regeneration.” Current Biology,22: 1166–1175.
(3) Español Suñer, R. et al. (2012) “Liver Progenitor Cells Yield Functional Hepatocytes in Response to Chronic Liver Injury in Mice.” Gastroenterology,143: 1564–1575.
(4) Zheng, D. et al. (2006) “Oval Cell Response in 2-Acetylaminofluorene/Partial Hepatectomy Rat Is Attenuated by Short Interfering RNA Targeted to Stromal Cell-Derived Factor-1.” The American Journal of Pathology,169: 2066–2074
A light activated (photoactivatable) tool for inducing liver damage was developed that applied a genetically-targeted photosensitizer system (5) to allow ablation of hepatocytes (LIVERZAP) (Figure 1A). LIVERZAP leads to rapid hepatocyte cell death in zebrafish embryos that is both non-invasive and less toxic than other available regeneration models. Zebrafish embryos are illuminated with the activating near-infrared light for only 12 minutes (A12min), which is sufficient to efficiently initiate the LIVERZAP ablation process (Figure 1B). Cell death first begins to occur 9 hours (hrs) after light treatment and is typically complete by 24hrs (R24hrs) after which the liver begins to regenerate (Figure 1C). During the injury process, the biliary epithelial cells (BECs) which form the biliary ductal network dramatically remodel in an attempt to maintain the architecture of the organ. However, LIVERZAP injury is sufficiently severe that the network collapses in its entirety by R24hrs (Figure 1C). Nevertheless, both hepatocyte number and the biliary network are restored and largely resemble unablated controls by 72hrs after light treatment (R72hrs). Fixed tissue analysis indicates that this repair process includes bursts of proliferation from hepatocytes, previously characterised progenitor-like cells (6,7) and BECs (Figure 1C). As the entire process of severe damage induction and regeneration takes only 72hrs using the LIVERZAP system, the key cell behaviours involved in both liver damage and repair can easily be observed and studied.
The extent of injury can be accurately controlled using light and hence can also be performed within a high-resolution microscope. In the project, a novel light-sheet microscopy approach was developed to visualise the cellular morphodynamics of the hepatic regenerative response in real-time after LIVERZAP-mediated liver damage. Detailed analysis of the time-lapse imaging data was performed by tracking of biliary epithelial cell (BEC) nuclei. These data revealed that both active migration and passive tissue growth contribute to liver regeneration. Bulk RNA-sequencing of hepatocytes and BECs has been performed. After in depth analysis and comparison to other published data sets, these data will be used to add mechanistic detail to the role of these regenerative cell behaviours.
A manuscript is currently being written to disseminate the results of the LIVERZAP project. Furthermore, the fellow has actively presented the work at several regional and international scientific conferences including the Annual Spring Meeting of the British Society for Developmental Biology (2019) and the Copenhagen Bioscience Conference, The Stem Cell Niche (2018). The fellow has taken part in a collaborative art-science dissemination project SciVi (http://www.scivi.dk/) with a view to publicising the research output of LIVERZAP to the lay public. Together with Berlin-based illustrator and live-painter Cosimo Miorelli , a short, animated video (or rather, a “speed-painted” video) of about 3.5 minutes will be produced (pending funding) to:
- present the practices of this specific research
- briefly explain how the liver works and why is it important
- show how the liver reacts to damage and regenerates (main focus of this research and of this video).
(5) He, J., et al. (2016) “A genetically targetable near-infrared photosensitizer.” Nature Methods, 13:263–268
(6) Lu, W.-Y. et al. (2015) “Hepatic progenitor cells of biliary origin with liver repopulation capacity.” Nature cell biology,17: 971–983.
(7) Choi, T.-Y. et al. (2014) “Extensive Conversionof Hepatic Biliary Epithelial Cells to Hepatocytes After Near Total Loss of Hepatocytes in Zebrafish.” Gastroenterology,146: 776–788
The extent of injury can be accurately controlled using light and hence can also be performed within a high-resolution microscope. In the project, a novel light-sheet microscopy approach was developed to visualise the cellular morphodynamics of the hepatic regenerative response in real-time after LIVERZAP-mediated liver damage. Detailed analysis of the time-lapse imaging data was performed by tracking of biliary epithelial cell (BEC) nuclei. These data revealed that both active migration and passive tissue growth contribute to liver regeneration. Bulk RNA-sequencing of hepatocytes and BECs has been performed. After in depth analysis and comparison to other published data sets, these data will be used to add mechanistic detail to the role of these regenerative cell behaviours.
A manuscript is currently being written to disseminate the results of the LIVERZAP project. Furthermore, the fellow has actively presented the work at several regional and international scientific conferences including the Annual Spring Meeting of the British Society for Developmental Biology (2019) and the Copenhagen Bioscience Conference, The Stem Cell Niche (2018). The fellow has taken part in a collaborative art-science dissemination project SciVi (http://www.scivi.dk/) with a view to publicising the research output of LIVERZAP to the lay public. Together with Berlin-based illustrator and live-painter Cosimo Miorelli , a short, animated video (or rather, a “speed-painted” video) of about 3.5 minutes will be produced (pending funding) to:
- present the practices of this specific research
- briefly explain how the liver works and why is it important
- show how the liver reacts to damage and regenerates (main focus of this research and of this video).
(5) He, J., et al. (2016) “A genetically targetable near-infrared photosensitizer.” Nature Methods, 13:263–268
(6) Lu, W.-Y. et al. (2015) “Hepatic progenitor cells of biliary origin with liver repopulation capacity.” Nature cell biology,17: 971–983.
(7) Choi, T.-Y. et al. (2014) “Extensive Conversionof Hepatic Biliary Epithelial Cells to Hepatocytes After Near Total Loss of Hepatocytes in Zebrafish.” Gastroenterology,146: 776–788
The novel regeneration model and imaging approach developed in the LIVERZAP project have huge potential to provide a detailed understanding of how cells behave to restore liver architecture after injury. The real time image data provided by this pipeline has already revealed the contribution of active migration of BECs to restoring the complex network of biliary ducts within the liver after severe injury. Further study will aim to use this system to better understand the mechanism by which this is achieved and to what degree this is essential for liver regeneration. While in this study the LIVERZAP model was used to study regeneration in zebrafish embryos due to their transparency, the system is currently being tested in adult zebrafish. Upon optimisation, LIVERZAP could therefore be used in combination with pharmaceutical screening to identify and develop potential therapeutics for the treatment of human liver diseases.