Lucy Erin O'Brien

Building epithelial architecture: insights from three-dimensional culture models

How do individual cells organize into multicellular tissues? Here, we propose that the morphogenetic behaviour of epithelial cells is guided by two distinct elements: an intrinsic differentiation programme that drives formation of a lumen-enclosing monolayer, and a growth factor-induced, transient de-differentiation that allows this monolayer to be remodelled.

Rac1 orientates epithelial apical polarity through effects on basolateral laminin assembly.

Cellular polarization involves the generation of asymmetry along an intracellular axis. In a multicellular tissue, the asymmetry of individual cells must conform to the overlying architecture of the tissue. However, the mechanisms that couple cellular polarization to tissue morphogenesis are poorly understood. Here, we report that orientation of apical polarity in developing Madin–Darby canine kidney (MDCK) epithelial cysts requires the small GTPase Rac1 and the basement membrane component laminin. Dominant-negative Rac1 alters the supramolecular assembly of endogenous MDCK laminin and causes a striking inversion of apical polarity. Exogenous laminin is recruited to the surface of these cysts and rescues apical polarity. These findings implicate Rac1-mediated laminin assembly in apical pole orientation. By linking apical orientation to generation of the basement membrane, epithelial cells ensure the coordination of polarity with tissue architecture.

Epithelial polarity and tubulogenesis in vitro

The most fundamental type of organization of cells in metazoa is that of epithelia, which comprise sheets of adherent cells that divide the organism into topologically and physiologically distinct spaces. Some epithelial cells cover the outside of the organism; these often form multiple layers, such as in skin. Other epithelial cells form monolayers that line internal organs, and yet others form tubes that infiltrate the whole organism, carrying liquids and gases containing nutrients, waste and other materials. These tubes can form elaborate networks in the lung, kidney, reproductive passages and vasculature tree, as well as the many glands branching from the digestive system such as the liver, pancreas and salivary glands. In vitro systems can be used to study tube formation and might help to define common principles underlying the formation of diverse types of tubular organ.

Morphological and biochemical analysis of Rac1 in three-dimensional epithelial cell cultures.

Rho GTPases are critical regulators of epithelial morphogenesis. A powerful means to investigate their function is three-dimensional (3D) cell culture, which mimics the architecture of epithelia in vivo. However, the nature of 3D culture requires specialized techniques for morphological and biochemical analyses. Here, we describe protocols for 3D culture studies with Madin-Darby Canine Kidney (MDCK) epithelial cells: establishing cultures, immunostaining, and expressing, detecting, and assaying Rho proteins. These protocols enable the regulation of epithelial morphogenesis to be explored at a detailed molecular level.

Beyond the Niche: Tissue-Level Coordination of Stem Cell Dynamics

Adult animals rely on populations of stem cells to ensure organ function throughout their lifetime. Stem cells are governed by signals from stem cell niches, and much is known about how single niches promote stemness and direct stem cell behavior. However, most organs contain a multitude of stem cell-niche units, which are often distributed across the entire expanse of the tissue. Beyond the biology of individual stem cell-niche interactions, the next challenge is to uncover the tissue-level processes that orchestrate spatial control of stem-based renewal, repair, and remodeling throughout a whole organ. Here we examine what is known about higher order mechanisms for interniche coordination in epithelial organs, whose simple geometry offers a promising entry point for understanding the regulation of niche number, distribution, and activity. We also consider the potential existence of stem cell territories and how tissue architecture may influence niche coordination.

Analysis of Membrane Traffic in Polarized Epithelial Cells

Spatial asymmetry is fundamental to the structure and function of most eukaryotic cells. A basic aspect of this polarity is that the cells plasma membrane is divided into discrete domains. The best studied and simplest example of this occurs in epithelial cells, which line exposed body surfaces. Epithelial cells use two pathways to send proteins to the cell surface. Newly made proteins can travel directly from the trans‐Golgi network (TGN) to either the apical or basolateral surface. Alternatively, proteins can be sent to the basolateral surface and then endocytosed and transcytosed to the apical surface. Epithelial cells grown on porous filters adopt a typical polarized morphology; transfected epithelial cells can be used to biosynthetically characterize the trafficking patterns of a given protein. These cells can also be used to study delivery to a particular surface and to localize the protein by immunofluorescence.

STAT1 Is Required for Redifferentiation during Madin-Darby Canine Kidney Tubulogenesis

Signal transducers and activators of transcription (STAT)1 is the key to the sequential control of Madin-Darby canine kidney tubulogenesis. Loss of STAT1 prevents redifferentiation. Constitutively active STAT1 is sufficient to restore cord formation but not mature lumens. These data suggest that STAT1 is necessary for the redifferentiation phase of tubulogenesis and that mature lumenogenesis requires a distinct signal.