Hepatoblasts are the bipotential progenitors of liver cells that differentiate into either hepatocytes or cholangiocytes. In their study, Marino Zerial and colleagues reveal how hepatoblast polarisation and lumen morphogenesis are linked to hepatocyte cell fate determination. To know more about their work, we spoke to the first authors, Maarten Bebelman and Lenka Belicova, and the corresponding author, Marino Zerial, Director and Scientific Member at the Max Planck Institute of Molecular Cell Biology and Genetics.
Maarten Bebelman, Lenka Belicova and Marino Zerial (left to right)
Marino, what questions is your lab trying to answer?
MZ: My lab has been investigating the molecular mechanisms of endocytosis, using a combination of light microscopy, quantitative image analysis, functional genomics and biochemical reconstitution. This work has led us to elucidate the role of mechanical forces in early endosome tethering and fusion. We are now applying these approaches to the study of liver tissue organisation, and its dysfunction in disease. In particular, we are interested in understanding the molecular mechanisms that decide the polarity of a hepatocyte and its response to luminal pressure. The particular cell architecture and polarity of hepatocytes contributes to the organisation of the liver tissue. It is this multi-scale perspective that fascinates us.
Maarten and Lenka, how did you come to work in the lab and what drives your research today?
LB: I joined the lab for my PhD. I was fascinated by the architecture of the liver Marino presented to me during the interview and wanted to understand how the cells become organised during development. I also started to work with a mammalian system for the first time and fell in love with the topic of tissue morphogenesis, on which I have continued to work during my postdoc.
MB: I joined Marino's lab as a postdoc with the idea of studying the endosomal system, the research topic that his lab is most known for. However, when I saw Lenka's PhD work on hepatocyte polarity and liver development, I realised that there were several fun and important questions that remained to be answered. So, I decided to start investigating how hepatocytes deal with, and respond to, bile fluid pressure during liver development.
Can you tell us about the background of the field that inspired your work?
LB: Cell fate decisions are coordinated at many levels and the role of mechanical and morphological cues is becoming more and more clear. For example, changes in cell shape can redistribute available factors, deform the nucleus, and spatially reorganise chromatin, leading to changes in gene expression. The establishment of apicobasal polarity and lumen formation leads to profound cellular changes and often coincides with cell differentiation. Epithelial cells in the liver have fascinated researchers over decades because unpolarised liver progenitors give rise to two cell types (hepatocytes and cholangiocytes) with remarkably different polarity (multi-polar versus uni-polar). These cells are, therefore, wonderful models to study the interplay of morphogenesis and cell fate decisions in development.
Can you give us the key results of the paper in a paragraph?
LB & MB: When liver progenitors differentiate into hepatocytes, they form small apical lumina between them and expand these anisotropically to generate a network of thin bile canaliculi (channels transporting bile) complementary to the sinusoidal network where blood flows. To do this, hepatocytes have a unique type of polarity, with multiple apical and basal surfaces. In contrast, when liver progenitors become cholangiocytes, they form a larger isotropic lumen, and the cells have a more common apicobasal polarity, with one apical and one basal surface. In our study, we investigated how differentiation of hepatoblasts into hepatocytes is affected if we prevent the anisotropic growth of their apical lumina and push them to display the common apicobasal polarity of cholangiocytes. Using various methods, we found that inducing isotropic lumen growth instead of anisotropic lumen elongation decreased the expression of hepatocyte markers and increased the expression of cholangiocyte-associated genes. This shows that lumen morphology feeds back to gene expression, and maintaining lumen anisotropy is required for proper hepatocyte differentiation. To our knowledge, this is the first evidence linking lumen morphology to the regulation of gene expression during differentiation of liver progenitors.
When doing the research, did you have any particular result or eureka moment that has stuck with you?
LB: In my PhD project, I aimed to understand how hepatocytes are structured, and one of the ways was to search for a ‘polarity switch’ that would change the unique polarisation phenotype of hepatocytes into that of a typical polarised epithelial cell. The discovery that hepatocytes deploy apical bulkheads as structural elements that ensure anisotropic lumen expansion was published in Journal of Cell Biology in 2021 (Belicova et al., 2021). As follow up, we assessed the effect of this polarity switch on hepatocyte differentiation, which developed into a full project. We had intense discussions on whether hepatocytes could sense their apical lumen and respond to changes in its shape. This seems counterintuitive because the predominant view is to consider the information flow from genes to changes in cellular morphology, and not the other way around. Of course, there is much more bi-directional feedback between gene expression and cell architecture, and it was exciting to uncover a feedback loop from cell organisation to gene expression. I hope this paper inspires other researchers to dig deeper and perhaps explore examples of such feedback in other systems.
MB: I got really excited when we found that reducing apical actomyosin contractility or increasing luminal pressure reduced the expression of typical hepatocyte genes and increased the expression of cholangiocyte-associated genes. It confirmed our hypothesis that apical lumen morphology influences hepatocyte differentiation during liver development.
Primary hepatoblasts in culture differentiate into hepatocytes under control conditions (left), forming bile canaliculi (green); upon Rab35 silencing (right), the cells arrange into cysts with an internal lumen reminiscent of cholangiocytes. The lateral marker E-cadherin (cadherin 1) is in magenta, and nuclei are in grey. Scale bars: 10 µm.
Primary hepatoblasts in culture differentiate into hepatocytes under control conditions (left), forming bile canaliculi (green); upon Rab35 silencing (right), the cells arrange into cysts with an internal lumen reminiscent of cholangiocytes. The lateral marker E-cadherin (cadherin 1) is in magenta, and nuclei are in grey. Scale bars: 10 µm.
And what about the flipside: any moments of frustration or despair?
MB: One of the methods we used in the paper to alter lumen morphology was silencing of the small GTPase Rab35. We hypothesised that the effect of Rab35 silencing on hepatoblast differentiation was due to its role in apical lumen morphology. To test this, we first tried a complex experiment to see if Rab35 knockdown in primary hepatoblasts could influence gene expression in neighbouring cells that had normal levels of Rab35. However, after half a year, we had to accept that this experiment was too technically challenging. Luckily, by then we had found that disrupting apical lumen morphology by reducing actomyosin contractility or increasing luminal pressure also affects hepatocyte differentiation, independently of Rab35 manipulation.
Why did you choose to submit this paper to Development?
LB: Development is one of my favourite journals, I read through each new issue and always find interesting papers. I believed that the story would be attractive to Development's readership, and I am glad my co-authors agreed.
Development is one of my favourite journals, I read through each new issue and always find interesting papers
MB: I agree with Lenka and would like to add that several key papers on the mechanisms of cell fate specification in the liver, which influenced our current work, were published in Development.
Maarten, what is next for you after this paper?
MB: I have recently moved to Utrecht University in The Netherlands, where I will conduct my postdoctoral research to investigate organelle trafficking in another model of cell polarity, neurons. I remain very interested in the intersection of cell biology, developmental biology, and biophysics, and I am convinced that I will further explore these fields in my future career.
Marino, where will this story take your lab next?
MZ: Capitalising on the mechanisms of hepatocyte polarity, we need to understand how changes in cell polarity regulate cell fate at the mechanistic level. The fact that hepatocytes respond to pressure in the bile canaliculi suggest the existence of mechano-sensing and -transduction mechanisms that govern the relationship between tissue architecture and cell differentiation. Understanding such mechanisms would provide the opportunity to gain insights into liver diseases that are characterised by alterations in luminal pressure, hepatocyte polarity and loss of metabolic pathways necessary for hepatocyte function.
Finally, let's move outside the lab – what do you like to do in your spare time?
LB: Besides research, I enjoy engaging in science outreach projects, for example creating content for the www.discoverliver.com website. When I need to disconnect completely from thinking about science, I like traveling with my family and friends, specifically somewhere where we can explore beautiful nature parks.
MB: In my spare time, I like to go out for long cycling trips. I especially enjoyed cycling through the beautiful hills surrounding Dresden.
M.B., L.B. & M.Z.: Max Planck Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany.
L.B.: Department of Cell and Molecular Biology, Karolinska Institutet, 171 77 Stockholm, Sweden.
E-mail: [email protected]
