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Teaser, summary, work performed and final results

Periodic Reporting for period 2 - RULLIVER (Rules of self-organization and reengineering of liver tissue)


Understanding the principles of tissue organization and their underlying molecular mechanisms remains an ambitious goal. For this, it is necessary to identify the quantitative rules that govern tissue organization and to link such rules to their underlying molecular mechanisms...


Understanding the principles of tissue organization and their underlying molecular mechanisms remains an ambitious goal. For this, it is necessary to identify the quantitative rules that govern tissue organization and to link such rules to their underlying molecular mechanisms to provide the means for reengineering the tissue. This requires monitoring of morphogenetic processes in space and time across different levels of complexity, from the molecular to the cellular, and from the cellular to the tissue scale. The pioneering work of Hans Elias in the 1950’s (Elias & Bengelsdorf, 1952) has established what remains today the textbook model of liver tissue organization. Since then, very little progress has been made towards improving the model. In addition, the model describes the tissue but does not make predictions for how the tissue can respond to perturbations.
We address this problem using the mouse liver as model system. The liver is a pertinent example of an organ with a complex 3D tissue organization. It consists of functional units, the liver lobule, containing two intertwined networks, the sinusoids for blood flow and the bile canaliculi (BC) for bile secretion and flux. Sinusoids and BC run antiparallel along the central vein (CV)-portal vein (PV) axis. The hepatocytes are the major parenchymal cells and display a peculiar and unique type of cell polarity distinct from that of simple epithelia. Whereas in epithelia all cells share the same orientation with their apical surface facing the lumen of the organ, hepatocytes are sandwiched between the sinusoidal endothelial cells and share the apical surface with multiple neighbouring hepatocytes to form a 3D BC network. Such an architecture makes it difficult to grasp the 3D organization of cells and tissue from 2D histological sections.
Our main aim is to understand the rules of self-organization of liver tissue and their implementation at the molecular level. Specifically, we plan to address the following questions: First, which are the rules whereby different cell types self-assemble to generate liver tissue? How are cell-cell interaction mechanics integrated with molecular signalling pathways to determine the tissue structure? Second, how can cell polarity be modified to generate the specific organization of hepatocytes? Hepatocytes share many of the molecular machineries expressed in polarized epithelial cells, including liver cholangiocytes, yet have a very different polarity. Therefore, which are the molecular principles responsible for shaping their topologically complex three-dimensional (3D) structure? Are these due to qualitative differences (e.g. hepatocyte-specific genes) or simply the result of quantitative tuning of the same machinery? Third, how are the basic cellular mechanisms, e.g. cell adhesion and cell-cell contacts, modified and/or tuned to generate the specific structure of liver tissue?
The ultimate goal will be to reengineer liver tissue structure as a means of validating our understanding. We will take advantage of a combination of properties of liver tissue to address fundamental questions concerning tissue organization. One of these properties is the tissue dynamics, whereby the liver constantly renews its cells and is capable of regeneration. Another interesting property of tissue organization is that not all hepatocytes are functionally identical within a lobule as different metabolic pathways show gradients of activity between PV and CV, a phenomenon termed metabolic zonation (Gebhardt & Hovhannisyan, 2010). Finally, we will apply newly available technologies to analyse and manipulate it both in vitro and in vivo make it an appropriate model of choice.

Work performed

Aim 1. In Aim 1 of this project, we worked on the development of a geometrical model of mouse liver tissue. This serves to generate an accurate 3D digital representation of the cells and their essential sub-cellular components in the developing, adult and regenerating mouse liver. We further developed our previously established technology (Morales-Navarrete et al. 2015; Morales-Navarrete et al., 2016; Wiegert et al., 2018), by establishing a pipeline consisting of an appropriate sample preparation, staining for ~20 organelle and pathway markers, microscopy imaging and new algorithms for image processing and analysis. We applied this pipeline to mouse liver tissue sections and used the images to develop and test the different algorithms required for the generation of the geometrical models. Using this pipeline, we investigated the formation of bile canaliculi (BC) in the developing liver through imaging of corresponding tissue samples with sub-cellular resolution. We could follow the polarization of hepatocytes, the expansion of the lumens and their connection leading to BC network formation. For investigations on the regenerating liver, we have been using the partial hepatectomy (70%) model (PH model). We focused on the role of cell division orientation in shaping the liver tissue. We found that, contrary to previous reports, hepatocytes divide in an oriented fashion and it is very likely that oriented cell division is crucial for the formation and orientation of the multi-cellular structural units (see below, Aim 4).
Aim 2. In Aim 2, we investigated the molecular mechanisms whereby hepatocytes are structured, and modify them to re-engineer cell polarity between the simple epithelial and hepatocyte organization. To this end, we needed to develop a variety of techniques to characterize epithelial vs. hepatocyte polarity, culture cells in vitro under conditions that recapitulate tissue features in vivo, and identify candidate genes and pathways accounting for cell fate decisions and cell polarization. We developed super-resolution Correlative Light Electron Microscopy (super-CLEM) method to map different fluorescent signals to the ultrastructure of cellular organelles (Franke et al., submitted). We developed new computational approaches to cluster genes on the basis of their multi-parametric signature. We established in vitro systems suitable to screen numerous genes and test them in corresponding assays. Thus, animal experiments are only necessary for the verification of promising candidates that we have identified in in vitro screens.
As a prerequisite for our investigations, we optimized our novel and unique in vitro cell culture system of primary foetal mouse hepatoblasts. Primary hepatoblasts in this cell culture system differentiate into hepatocytes and recapitulate formation of the bile canaliculi, whereas common epithelial cells, e.g., primary bile duct cells, establish 3D cyst structures with a shared hollow lumen. Primary hepatocytes are furthermore known to lose cell polarity and hepatocellular function within the first 24 hours after isolation and when plated under conventional monolayer culture conditions. However, in our system, these cells recapitulate basic aspects of tissue organization. We performed a focused screen of a set of ~120 genes in total. We were able to identify two genes, previously unrelated to hepatocyte polarity, whose silencing resulted in a switch from the BC towards the common epithelial cyst-like lumen morphology. Preliminary data indicate that the two candidates operate via a joint pathway, but validation experiments are still ongoing.
Aim 3. Based on the results of Aim 1 and Aim 2, we introduce genetic or pharmacological perturbations in vivo to reengineer the structure and function of liver tissue. We first sought to establish a method for silencing genes in the mouse embryonic liver using a mouse strain ubiquitously expressing Green Fluorescent Protein GFP. Through the application of inno

Final results

Through the use of quantitative image analysis and multi-scale phenotypic analysis, we succeeded in developing a state-of-the art 3D model that can make predictions not only how liver tissue is built but also modified in response to genetic perturbations. The conceptual and technological advances obtained will increase our understanding of human liver diseases, currently a major unmet biomedical need. As proof-of-principle we succeeded in re-engineering liver tissue as one of the main aims of this proposal. Using different approaches, we were able to identify candidate genes, previously unrelated to hepatocyte polarity, and to modify cell polarity between the simple epithelial and hepatocyte organization.
Three cutting edge technologies enable this project: First, we have developed a unique cell culture system that enables us to cultivate primary liver cells (hepatoblasts and hepatocytes) in vitro in such a way that they can recapitulate basic aspects of tissue organization. Second, the vertebrate liver is uniquely suited to functional genomics: we are able to silence genes potently and specifically both in primary cells and in the embryonic and adult liver in vivo. Third, we have been developing new image processing and analysis algorithms to extract quantitative information from liver tissue and integrate omics.

Website & more info

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