UC San Diego

Many vertebrate organ systems contain a specialized ciliated epithelium decorated with motile cilia, which produce a ciliary flow in order to move mucus or fluid across the tissue surface. Examples include the proximal airways of the respiratory tract, oviduct, ependyma of the brain ventricles and the embryonic node. The importance motile cilia function in these specialized tissues to organ function is evident from human diseases such as primary ciliary dyskinesia (PCD) or immotile cilia syndrome. Patients with PCD are prone to chronic respiratory infections, sinus infections, in 50% of cases have situs inversus and in rare cases exhibit hydrocephalus. This broad array of phenotypes due to ciliary dysfunction clearly shows the importance of ciliated epithelia to organismal survival. A great deal is known about ciliary flow and how it relates to tissue function in relation to disease states. However, much less is known about how such tissues are formed during development, and what factors control the formation of cilia in these tissues. In this work I first describe the morphogenesis of the ciliated external epithelium of Xenopus laevis embryos, a tissue that closely resembles the respiratory epithelium in form and function. Cells with motile cilia cover the Xenopus embryo in a characteristic spacing pattern. This pattern arises early in development when cells in the inner layer of the ectoderm are selected by Notch signaling to form ciliated cell precursors (CCPs) that then undergo radial intercalation into the outer epithelial layer to form ciliated cells. Inhibition of Notch signaling results in an overproduction of CCPs; while radial intercalation becomes limiting ciliated cells maintain their spacing in the epithelium. Transgenic and transplantation assays to mark ciliated cells and intercalating populations, respectively, indicate that intercalating cells are free to wedge basolaterally, but can only insert apically at vertices where multiple outer cells make contact, likely making apical insertion the rate-limiting step during radial intercalation. Ciliated cell spacing also appears to be influenced by several other factors including competition with intercalating non-ciliated cell populations for vertices, cell morphology, and limitations on apical insertion likely imposed by the outer layer. Suggesting that cells other than the ciliated cells themselves can affect the ciliated cell spacing pattern, and thus final tissue architecture observed in such an epithelium. In the second part of this dissertation I describe a role for FoxJ1, a forkhead transcription factor, in the specification of node-like cilia in Xenopus and zebrafish embryos. Monociliate cells at the embryonic node generate a leftward fluid flow responsible for left-right asymmetry breaking in mouse, fish and Xenopus embryos. These cilia share features of both primary sensory cilia and motile cilia found on multiciliate cells, but how these cilia are specified in relation to other cilia is unknown. Using knockdown by morpholino injection, I show that FoxJ1 plays a conserved role in basal body docking in multiciliate cells found on the external epithelium of Xenopus embryos. However, in contrast to results in the mouse, I also show that FoxJ1 is required for formation of the node-like cilia in Xenopus gastrocoel roof plate (GRP) and zebrafish Kupffer's vesicle. Additionally, I show that misexpression of FoxJ1 is sufficient to induce ectopic GRP -like cilia on the surface epithelial cells of Xenopus embryos. Microarray analysis further indicates that FoxJ1 can induce ectopic cilia formation by upregulating the expression of genes required for cilia structure as well as genes required for cilia motility. Together these results indicate that node-like cilia in Xenopus and zebrafish are likely generated using a genetic pathway similar to that used to specify cilia in multiciliate cells. The studies presented in this dissertation shed light on the both the morphogenetic events that underlies the formation of the ciliated external epithelium in Xenopus embryos, but also on the specification of two different cilia subtypes that form on the epithelia of developing Xenopus embryo