Building a ciliated epithelium: Transcriptional regulation and radial intercalation of multiciliated cells

Abstract

The epidermis of the Xenopus embryo has emerged as a powerful tool for studying the development of a ciliated epithelium. Interspersed throughout the epithelium are multiciliated cells (MCCs) with 100 + motile cilia that beat in a coordinated manner to generate fluid flow over the surface of the cell. MCCs are essential for various developmental processes and, furthermore, ciliary dysfunction is associated with numerous pathologies. Therefore, understanding the cellular mechanisms involved in establishing a ciliated epithelium are of particular interest. MCCs originate in the inner epithelial layer of Xenopus skin, where Notch signaling plays a critical role in determining which progenitors will adopt a ciliated cell fate. Then, activation of various transcriptional regulators, such as GemC1 and MCIDAS, initiate the MCC transcriptional program, resulting in centriole amplification and the formation of motile cilia. Following specification and differentiation, MCCs undergo the process of radial intercalation, where cells apically migrate from the inner layer to the outer epithelial layer. This process involves the cooperation of various cytoskeletal networks, activation of various signaling molecules, and changes in cell-ECM and cell-cell adhesion. Coordination of these cellular processes is required for complete incorporation into the outer epithelial layer and generation of a functional ciliated epithelium. Here, we highlight recent advances made in understanding the transcriptional cascades required for MCC specification and differentiation and the coordination of cellular processes that facilitate radial intercalation. Proper regulation of these signaling pathways and processes are the foundation for developing a ciliated epithelium.

Introduction

Ciliated epithelia are important in a wide variety of physiological contexts, where they function to generate fluid flow over the surface of various tissues. In mammals, these epithelia are found in the airway to help mucus flow over the surface, the ependyma that line the brain ventricles to move cerebral spinal fluid, and the oviduct where they facilitate ovum transport. Fluid flow is generated by highly specialized multiciliated cells (MCCs) that contain 100 + cilia that beat in a coordinated and polarized manner to generate the directed fluid flow over the surface (Brooks & Wallingford, 2014; Choksi, Lauter, Swoboda, & Roy, 2014). Importantly, defects in the generation or maintenance of a ciliated epithelium are found in many pathologies, as ciliary dysfunction and ciliopathies are central to many diseases (Hildebrandt, Benzing, & Katsanis, 2011; Reiter & Leroux, 2017). Therefore, there is significant interest in understanding the molecular events and signaling pathways that regulate MCC specification, differentiation, and function.

The epidermis of the Xenopus embryo has emerged as a powerful model system for studying the development and maintenance of a ciliated epithelium (Walentek & Quigley, 2017; Werner & Mitchell, 2013). Although this epithelium is derived from the ectoderm in Xenopus rather than the endoderm, the transcriptional cascades that govern the formation of this tissue, as well as its morphogenesis, are highly conserved with mammalian lung tissue (Brooks & Wallingford, 2014; Walentek & Quigley, 2017). Recent single cell RNA sequencing in Xenopus has provided us with a powerful tool to trace MCC differentiation in the developing vertebrate embryo. This targeted tracing revealed that differentiation of MCCs begins as early as stage 11 of development when MCC genes including forkhead box protein J1 (Foxj1), myb, forkhead box protein n4 (Foxn4), and regulatory factor X2 (Rfx2) become enriched in cells that will develop into ciliated epidermal progenitors (Angerilli, Smialowski, & Rupp, 2018; Briggs et al., 2018). Activation of various transcriptional pathways and signaling networks within ciliated cell precursors commits these cells to an MCC lineage and establishes a precise transcriptional cascade that drives MCC differentiation.

A significant body of work has contributed to our understanding of the process of MCC specification and differentiation, but it was not initially known where these processes occurred. The Xenopus skin is multilayered, leading to the possibility that MCCs are either (1) derived from cells in the most superficial layer of the ectoderm where they are found in their mature state, or (2) derived from a deeper layer, requiring migration to the outer epithelium. In order to test these possibilities, transplantation studies were performed in which labeled outer epithelial cells or labeled deep ectodermal cells (below the outer layer) were transplanted onto unlabeled embryos during mid-gastrulation. These classic transplantation and tracing studies revealed that MCC specification and differentiation occurs within a deeper layer of the Xenopus skin and is followed by a short, but directed, apical migration into the outer epidermis, a process termed radial intercalation (Drysdale & Elinson, 1992). Of note, the early transplantation studies revealed that another cell type, later identified as ionocytes (ICs), intercalates alongside of MCCs (Drysdale & Elinson, 1992; Quigley, Stubbs, & Kintner, 2011), and later studies revealed that a third cell type, small secretory cells (SSCs), also undergoes radial intercalation (Dubaissi et al., 2014).

Following the intercalation of multiple cell populations, the mature ciliated epithelium is comprised of the following cell types: secretory cells that release mucus (e.g., Goblet cells in mammals and mucus-secreting cells in Xenopus) and antimicrobial substances (e.g., Club cells in mammals and SSCs in Xenopus), MCCs that generate fluid flow and distribute the released substances over the surface, and ICs which play a role in ion regulation (Dubaissi & Papalopulu, 2011; Dubaissi et al., 2014; Haas et al., 2019). The development of the Xenopus ciliated epithelium at the external surface provides a useful tool for studying the development of this highly specialized and complex tissue, as well as providing a model for studying mucociliary diseases (Nenni et al., 2019; Walentek & Quigley, 2017).

There have been several recent reviews that address different aspects of ciliated epithelial development including centriole amplification and cilia polarity (Boutin & Kodjabachian, 2019; Brooks & Wallingford, 2014; Lewis & Stracker, 2020; Zhang and Mitchell, 2015a, Zhang and Mitchell, 2015b). Here, we will focus on our current understanding of the transcriptional networks required for MCC specification and differentiation and the process of MCC radial intercalation in the Xenopus epidermis, which are required for the formation of a functional ciliated epithelium.