Anisotropic expansion of hepatocyte lumina enforced by apical bulkheads

Abstract

Lumen morphogenesis is key to the function of organs and results from the integration of molecular pathways and mechanical forces. The mechanisms governing anisotropic lumen expansion remain elusive. In contrast to epithelial cells which have simple apico-basal polarity and form tubes, hepatocytes are multi-polar and form narrow lumina that grow anisotropically between adjacent cells, collectively generating a complex 3D network of bile canaliculi (BC). Here, we studied lumen elongation and BC morphogenesis in differentiating primary mouse hepatoblasts in vitro. Remarkably, we discovered a pattern of specific extensions of the apical membrane traversing the lumen between adjacent hepatocytes and sealed by tight junctions, reminiscent of the bulkheads of boats. These structures were also present in the developing liver. A targeted screen revealed that silencing of Rab35 caused loss of the bulkheads, conversion of hepatocyte into simple epithelial polarity and formation of spherical lumina in vitro. Strikingly, we could re-engineer hepatocyte polarity and tissue morphogenesis in vivo in the embryonic liver, converting BC into simple epithelial tubes. Our results suggest that the apical bulkheads of hepatocytes are cell-intrinsic anisotropic mechanical elements that ensure stability of the elongating lumen between two cells, thus determining the structure of BC during liver tissue morphogenesis.

Rab8, Rab11, and Rab35 coordinate lumen and cilia formation during Zebrafish Left-Right Organizer development

“…Previous work in mammalian culture systems and with preliminary morpholino studies in zebrafish KV have implicated Rab8, Rab11, and Rab35 in cilia and/or lumen establishment (Bryant et al ., 2010; Knödler et al ., 2010; Westlake et al ., 2011; Klinkert et al ., 2016; Kuhns et al ., 2019; Naslavsky and Caplan, 2020; Belicova et al ., 2021). However, their cellular distribution during KV development has not been investigated, nor has it been positioned in relation to KV cilia formation or to actin recruitment to the apical membrane.…”

“…Previous work in mammalian culture systems and with preliminary morpholino studies in zebrafish KV have implicated Rab8, Rab11, and Rab35 in cilia and/or lumen establishment (Bryant et al, 2010;Knödler et al, 2010;Westlake et al, 2011;Klinkert et al, 2016;Kuhns et al, 2019;Naslavsky and Caplan, 2020;Belicova et al, 2021).…”

“…These screens involve the use of mammalian cell culture systems that are either depleted or genetically null for Rab GTPase family members (Yoshimura et al ., 2007; Oguchi et al ., 2020; Homma et al ., 2021). Some Rab GTPases identified were involved in either cilia or lumen formation, and a subset of those were involved in both such as Rab11, Rab8, and Rab35 (Bryant et al ., 2010; Knödler et al ., 2010; Westlake et al ., 2011; Klinkert et al ., 2016; Kuhns et al ., 2019; Naslavsky and Caplan, 2020; Belicova et al ., 2021). Here we investigated these three Rab GTPases in KV development.…”

Three-dimensional Characterization of Interorganelle Contact Sites in Hepatocytes using Serial Section Electron Microscopy

“…To better understand liver biology, this approach can be used to investigate hepatocyte morphology in 2D cell culture, 3D hepatic organoid model systems 49 , and liver tissue. This robust and flexible approach can be used by many laboratories with access to a conventional 120 kV transmission electron microscope and ultramicrotome and does not require expensive sample preparation techniques.…”

Hepatoblast iterative apicobasal polarization is regulated by extracellular matrix remodeling

“…Later, the lumina open and expand due to secretion of osmotically active molecules by the hepatoblasts (Dasgupta, 2018 15 ). The peculiarity of bile canalicular expansion is its extreme anisotropy, which is enforced by mechanical elements named apical bulkheads (Belicova, 2021 16 ; Bebelman 2023 17 ). This expansion results in the formation of narrow tubes.…”