MCC differentiation is orchestrated by a coordinated cascade of regulators, including signaling pathways and transcription factors (Figure 1). Previous studies have demonstrated that the inhibition of the Notch and bone morphogenic protein (BMP) signaling pathways triggers a regulatory network of genes involved in the MCC fate determination of developing epithelia [30,31,32,33]. The upstream mechanisms that repress the Notch and BMP pathways are not fully understood. However, the microRNA clusters miR-34/miR-449 have been shown to play essential roles at multiple levels in multiciliogenesis by destabilizing target mRNAs [34,35]. The miR-34/miR-449 family includes the functionally redundant miRNA clusters miR-34b/c and miR-449a/b/c, which are highly expressed in multiciliated tissues. In particular, miR-449 has been shown to promote MCC differentiation by directly repressing Notch1 and its ligand Delta-like 1 (DLL1) in progenitor cells [35,36,37].

Downstream of Notch inhibition, the MCC differentiation gene regulatory network is controlled by the coiled-coil Geminin family proteins Geminin (GMNN), Geminin coiled-coil domain-containing protein 1 (GEMC1), and MCIDAS (also known as multicilin or IDAS) [38,39,40,41,42,43,44,45,46]. Both GEMC1 and MCIDAS are master regulatory proteins that are necessary and sufficient for MCC differentiation [43,47]. Upon forming a ternary complex with the cofactor DP1 and transcription factors E2F4/5, GEMC1 is able to activate expression of MCIDAS and other downstream ciliary genes. MCIDAS also acts as a transcriptional activator by forming a complex with DP1 and E2F4/5 to stimulate gene expression essential for centriole biogenesis. Early multiciliogenesis factors that are downstream of GEMC1 and MCIDAS include deuterosome assembly protein 1 (DEUP1), MYB, cyclin O (CCNO), forkhead box J1 (FOXJ1), and RFX2/3 [41,43,44,45,48]. p73, another transcription factor downstream of MCIDAS, was found to activate expression of FOXJ1, RFX2/3, and miR-34b/c and nearly 50 other ciliary genes in mouse tracheal MCCs [49,50]. While the role of Geminin in MCC differentiation is less understood, it has been shown to act as an inhibitor of GEMC1 and MCIDAS function [40,44].

During MCC differentiation, the biogenesis of hundreds of centrioles occurs through two different pathways: the canonical centriolar pathway, which utilizes pre-existing centrioles for new centriole generation, and the de novo deuterosome-mediated pathway [51,52]. Although a small fraction of centrioles is produced via the canonical centriolar pathway, the majority are thought to be amplified from deuterosomes. Deuterosomes are electron-dense, fibrogranular structures that contain several proteins required for centriole amplification, such as DEUP1 and coiled-coil domain-containing 78 (CCDC78) [48,53,54]. Although research on the origins and molecular components of deuterosomes is still ongoing, it has been reported that deuterosomes are nucleated from existing centrioles or spontaneously synthesized in the cytoplasm [55,56,57]. A recent study questioned the need for the deuterosome in MCC centriole amplification, since MCCs lacking deuterosomes are able to amplify the correct number of centrioles with normal kinetics [58].

Following release from the deuterosome or centrosomal centriole, the nascent centrioles migrate to the apical cell surface, dock at the plasma membrane, and mature into basal bodies to initiate cilium assembly. For efficient basal body docking to occur, small vesicles are recruited to the distal appendages of centrioles and then fuse to form a larger membranous cap called the ciliary vesicle [13,53,59]. Subsequently, the ciliary vesicle flattens and forms a bilayer membranous sheath around the developing ciliary axoneme that merges with the cell membrane, allowing the elongating cilium to protrude from the apical cell surface. Vesicular trafficking during ciliogenesis has been studied mainly using mammalian cultured cells with primary cilia. In early stages of ciliogenesis, the fusion of small vesicles into the ciliary vesicle is mediated by the EPS-15 homology domain-containing (EHD) family of membrane-shaping proteins, EHD1 and EHD3 [52,60]. The small GTPase Rab11 then recruits and activates Rabin8, a guanine nucleotide exchange factor (GEF) for Rab8, in the vicinity of the centrosome. Rabin8, in turn, promotes the recruitment and local activation of Rab8 to facilitate ciliary membrane growth and vesicular transport of ciliary proteins into the cilium [61,62]. Rab8 and Rabin8 also interact with the distal appendage protein CEP164 (also known as NPHP15), which acts as a molecular bridge between the mother centriole and components of the ciliary membrane assembly machinery [12]. Our group demonstrated that, in airway MCCs, the 15-kDa coiled-coil protein Chibby1 (Cby1) plays an important role in ciliary vesicle formation and basal body docking (Figure 2) [13,63]. Cby1^−/− mice exhibit chronic upper airway infection, due to a markedly decreased number of multicilia and a complete absence of mucociliary clearance activity [63]. Cby1 and its interacting proteins localize to the base of cilia (Figure 2A). Cby1 is recruited to the distal appendages of basal bodies through its physical interaction with CEP164 and interacts with Rabin8 to facilitate the efficient assembly of ciliary vesicles (Figure 2B) [13,64]. Cby1 exists in a complex with the membrane remodeling proteins, Chibby1-interacting Bin/Amphiphysin/Rvs (BAR) domain-containing 1 and 2 (ciBAR1 and ciBAR2; previously known as FAM92A and FAM92B, respectively) [64,65]. Coexpression of ciBAR1 or ciBAR2 with Cby1 in mammalian cultured cells induces the formation of globular and tubular membrane structures, suggesting that the Cby1/ciBAR complex facilitates ciliogenesis through regulation of membrane-remodeling processes.

The docking and planar polarization of basal bodies are controlled by the interplay between the planar cell polarity (PCP) pathway and the underlying actin and microtubule cytoskeleton [66,67,68,69,70,71]. The PCP pathway is a noncanonical Wnt pathway and has emerged as a critical regulator of MCC differentiation. In MCCs, the orientation of basal bodies is established via rotational polarity at the apical cell surface to enable synchronous beating of motile multicilia on each cell. PCP components localize to opposing membrane domains at the proximal and distal sides of cells [68,69]. The core PCP components include the evolutionarily conserved transmembrane proteins Frizzled, Van Gogh-like (Vangl, Van Gogh in Drosophila), Celsr (Flamingo in Drosophila) and cytoplasmic proteins Dishevelled, Prickle, and Inversin/Diversin (Diego in Drosophila). Mutations in the core PCP components lead to basal body docking defects and random orientation of basal bodies and ciliary beating in MCCs.

The extension and maintenance of cilia depend on the IFT machinery [18,19]. Along the cilium, anterograde IFT movement is mediated by kinesin-2 motors (from base to tip), whereas retrograde transport is powered by cytoplasmic dynein-2 (from tip to base). Fully developed cilia are highly dynamic and constantly undergo turnover with continuous transport of proteins and lipids in and out of cilia [72,73].

The male reproductive tract is composed of several highly convoluted tubular segments, each of which plays a crucial role in the development of fully functional spermatozoa (sperm) (Figure 3A). The testis, consisting of long convoluted seminiferous tubules, is the site of spermatogenesis [74,75,76]. From the testis, the spermatozoa are collected in the rete testis and are transported into the epididymis via the efferent ducts (EDs) [77,78]. The EDs contain specialized MCCs that prevent sperm from aggregating [79]. In the epididymis, sperm continue to mature and are finally stored in the cauda epididymis for eventual release via the vas deferens [80,81].

The development of spermatozoa takes place in the testis. The seminiferous tubules consist of multiple cell types, including spermatogenic cells, Sertoli cells, and peritubular myoid cells. The spermatogenic cycle occurs in waves, and different regions of the seminiferous tubules contain spermatogenic cells at varying steps of differentiation. Sertoli cells are nurse cells that aid spermatogenesis by anchoring developing spermatids to the seminiferous tubule epithelium and establishing the blood–testis barrier [82]. Peritubular myoid cells are smooth muscle-like cells that surround the seminiferous tubules and provide structural rigidity to the seminiferous tubules and contract to push spermatozoa and fluid into the rete testis.

Spermatogenesis is initiated by the asymmetric cell division of spermatogonial stem cells. Following several rounds of mitotic cell divisions, primary spermatocytes go through two rounds of meiosis to give rise to spermatids [75]. Spermatids then undergo spermiogenesis, in which round spermatids transform into elongated spermatozoa with a flagellum and a species-specific head shape [74]. Upon completion of spermiogenesis, the spermatozoa appear morphologically mature but lack motility and the capacity to fertilize an egg [83]. Sertoli cell secretions release spermatozoa into the lumen of the seminiferous tubules and coordinated contractions of peritubular myoid cells propel sperm into the rete testis [79].

Once collected in the rete testis, sperm transit into the EDs. The EDs originate from mesonephric tubules and are critical for concentrating the seminiferous fluid by reabsorbing water and ions and transporting sperm into the epididymis [78,84,85,86]. Three-dimensional reconstruction of mouse EDs revealed that EDs are 22.5 µm in mean radius and 81.9 mm in length [87]. Sperm transport through the EDs occurs within 45 min in rats [88]. The number of EDs in the mice varies from 2 to 5 [87,89,90,91]. In golden Syrian hamsters, there are six ducts [92]. In humans and other large mammals, EDs are more numerous and feed into the epididymis at various sites, whereas EDs in rodents collect into a singular duct (known as the common duct) prior to entering the epididymis [93].