Mouse Fetal Whole Intestine Culture System for Ex VivoManipulation of Signaling Pathways and Three-dimensional Live Imaging of VillusDevelopment

摘要

Most morphogenetic processes in the fetal intestine have been inferred from thin sections of fixed tissues, providing snapshots of changes over developmental stages. Three-dimensional information from thin serial sections can be challenging to interpret because of the difficulty of reconstructing serial sections perfectly and maintaining proper orientation of the tissue over serial sections. Recent findings by Grosse et al., 2011 highlight the importance of three- dimensional information in understanding morphogenesis of the developing villi of the intestine¹. Three-dimensional reconstruction of singly labeled intestinal cells demonstrated that the majority of the intestinal epithelial cells contact both the apical and basal surfaces. Furthermore, three-dimensional reconstruction of the actin cytoskeleton at the apical surface of the epithelium demonstrated that the intestinal lumen is continuous and that secondary lumens are an artifact of sectioning. Those two points, along with the demonstration of interkinetic nuclear migration in the intestinal epithelium, defined the developing intestinal epithelium as a pseudostratified epithelium and not stratified as previously thought¹. The ability to observe the epithelium three-dimensionally was seminal to demonstrating this point and redefining epithelial morphogenesis in the fetal intestine. With the evolution of multi-photon imaging technology andthree-dimensional reconstruction software, the ability to visualize intact, developingorgans is rapidly improving. Two-photon excitation allows less damaging penetration deeperinto tissues with high resolution. Two-photon imaging and 3D reconstruction of the wholefetal mouse intestines in Walton et al., 2012 helped to define thepattern of villus outgrowth². Here we describe a whole organ culture systemthat allows ex vivo development of villi and extensions of that culturesystem to allow the intestines to be three-dimensionally imaged during theirdevelopment.