Planar cell polarity pathway as a master regulator of biliary morphogenesis

Abstract

The biliary tree is an intricate network of ducts within the liver, responsible for transporting bile to the intestine. The development of a functional biliary system during organogenesis is indispensable to mammalian health and abnormal bile duct formation leads to paediatric cholestatic diseases such as biliary atresia and Alagille syndrome. While such human genetic conditions and animal research have identified the role of key molecular regulators, such as the Notch and TGFβ signaling pathways, in determining biliary lineage, the mechanisms driving the morphological transformation of a simple ductal plate into a complex tubular network remain largely elusive. Here, I aimed to identify candidate regulators of terminal morphogenesis in bile duct development and functionally define the molecular mechanisms that underpin this process. Analysis of a single-cell RNA-sequencing dataset of embryonic mouse livers revealed increased transcriptional expression of Planar Cell Polarity (PCP) pathway components during terminal biliary development. This increase in expression coincides with the timeframe during which the ductal plate undergoes significant morphological reorganisation. Using a combination of transgenic and mutant mouse lines and 3D organoid cultures, I demonstrated that PCP signaling is essential for proper bile duct morphogenesis. The use of whole-mount immunofluorescence and proteomics revealed that VANGL2, a core PCP component, localised to cell-cell junctions of ductular cells and interacts with the desmosome complex. Consequently, the genetic loss of Vangl2 function led to the universal disruption of cell-adhesions between ductular cells suggesting a novel role for PCP in supporting the functional assembly of cell-cell junctions. Moreover, this also resulted in failure to establish a continuous biliary network, with mutant animals displaying fewer bile duct branches that remained disconnected, along with defects in terminal bile duct differentiation. Additionally, mutant bile ducts demonstrated aberrant apical-basal cell polarity as evidenced by: (i) disrupted cytoskeletal arrangements, and (ii) impaired efflux pump activity, thus highlighting the essential link between normal tissue morphology and bile duct physiology. In this thesis I provide one of the first mammalian descriptions of the biomechanical regulators of bile duct morphogenesis.