DEVELOPMENT 2507 RESEARCH ARTICLE INTRODUCTION Cells with beating cilia are a common feature of many organ systems that depend on a directed fluid flow to function (Afzelius, 1995). For example, ciliated cells produce fluid flow in tissues as diverse as: the respiratory tract of mammals, where they clear mucous and debris; the choroid plexus, where they circulate the cerebral spinal fluid into the ventricles of the brain; and the reproductive tract, where they transport the egg along the oviduct. Proper development and function of these organs, therefore, requires the formation of a specialized epithelium containing cells with motile cilia. The skin of the amphibian embryo also produces a directed fluid flow generated by ciliated cells, thus serving as a model system for studying how such ciliated epithelia form during organogenesis. In Xenopus, the skin develops after gastrulation through the differentiation of two cell types that are derived from two distinct layers of the ectoderm (Fig. 1A) (Drysdale and Elinson, 1992). Cells in the outer layer of the ectoderm, also called the superficial layer, differentiate into mucus-producing epidermal cells, thus forming an occluding epithelial barrier on the embryo surface. Cells in the inner layer of the ectoderm, also called the sensorial layer, spread out underneath the outer layer during epiboly (Keller, 1980) and a subset give rise to ciliated cell precursors (CCPs) during early neurulae stages (stages 12-14) (Deblandre et al., 1999; Drysdale and Elinson, 1992). These precursors then differentiate into ciliated cells by intercalating radially into the outer layer during mid neurulae stages (stages 16-20) and undergoing ciliogenesis, allowing them to produce a directed fluid flow by late neurulae stages (stages 22-26). Ciliated cell differentiation is precisely controlled, thus ensuring that the cells are distributed across the epidermal surface at high density in an evenly spaced pattern. In many developing tissues, specific spacing patterns of differentiated cells are generated by lateral inhibition, an evolutionarily conserved process in which cells inhibit their neighbors from acquiring the same fate using the Notch signaling pathway (Kintner, 2003). Studies of Notch in the Xenopus skin indicate that lateral inhibition also operates during the formation of ciliated cells, whereby Notch negatively regulates the number of CCPs that form in the inner layer of the ectoderm (Deblandre et al., 1999). By determining CCP number, the process of lateral inhibition could conceivably act to distribute ciliated cells evenly across the skin surface. However, when Notch is inhibited and CCPs are overproduced, the density of ciliated cells detected at tadpole stages only increases about twofold; moreover, these cells remain spaced out (Deblandre et al., 1999). Thus, although Notch determines the number of CCPs that form in the inner layer, other factors determine the pattern of ciliated cells in the outer layer. As CCPs need to intercalate radially to become ciliated cells, one possibility is that this morphogenetic process is a crucial step in controlling the pattern of ciliated cell differentiation (Deblandre et al., 1999). By marking inner and outer cells with lineage tracers, Drysdale and Elinson (Drysdale and Elinson, 1992) showed that inner cells contribute not only ciliated cells but also an equal population of intercalating non-ciliated cells (INCs) to the outer layer (Fig. 1A). Thus, the pattern of CCPs in the outer layer may not only be determined by their ability to intercalate but also by interactions with the INCs. To examine these issues, we used two assays to characterize inner cells during radial intercalation. We first show using a transplantation assay that inhibiting Notch leads to more CCs and INCs in the outer layer, although their number and distribution differ significantly. We then develop a transgenic assay to distinguish CCPs from INCs in order to describe the behavior of these two cell types during intercalation both normally and when they are overproduced after disabling Notch signaling. The results of these analyses reveal important morphological differences between CCPs and INCs at the earliest stages of radial intercalation. We propose that these differences, along with limitations imposed on intercalation at the apical surface by the outer layer determine the pattern of ciliated cells found in the Xenopus skin. Radial intercalation of ciliated cells during Xenopus skin development Jennifer L. Stubbs 1,2 , Lance Davidson 3, *, Ray Keller 3 and Chris Kintner 1,† Cells with motile cilia cover the skin of Xenopus tadpoles in a characteristic spacing pattern. This pattern arises during early development when cells within the inner layer of ectoderm are selected out by Notch to form ciliated cell precursors (CCPs) that then radially intercalate into the outer epithelial cell layer to form ciliated cells. When Notch is inhibited and CCPs are overproduced, radial intercalation becomes limiting and the spacing of ciliated cells is maintained. To determine why this is the case, we used confocal microscopy to image intercalating cells labeled using transplantation and a transgenic approach that labels CCPs with green fluorescent protein (GFP). Our results indicate that inner cells intercalate by first wedging between the basal surface of the outer epithelium but only insert apically at the vertices where multiple outer cells make contact. When overproduced, more CCPs are able to wedge basally, but apical insertion becomes limiting. We propose that limitations imposed by the outer layer, along with restrictions on the apical insertion of CCPs, determine their pattern of radial intercalation. KEY WORDS: Ciliated cells, Intercalation, Epithelium Development 133, 2507-2515 (2006) doi:10.1242/dev.02417 1 Salk Institute for Biological Studies, Molecular Neurobiology Laboratory, La Jolla, CA 92037, USA. 2 Division of Biology, University of California San Diego, La Jolla, CA 92037, USA. 3 University of Virginia, Department of Biology, Charlottesville, VA 22905, USA. *Present address: Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA 15260, USA † Author for correspondence (e-mail: kintner@salk.edu) Accepted 26 April 2006