A function for the Joubert syndrome protein Arl13b in ciliary membrane extension and ciliary length regulation.

Cilia perform a variety of functions in a number of developmental and physiological contexts, and are implicated in the pathogenesis of a wide spectrum of human disorders. While the ciliary axoneme is assembled by intraflagellar transport, how ciliary membrane length is regulated is not completely understood. Here, we show that zebrafish embryos as well as mammalian cells overexpressing the ciliary membrane protein Arl13b, an ARF family small GTPase that is essential for ciliary differentiation, showed pronounced increase in ciliary length. Intriguingly, this increase in cilia length occurred as a function of the amounts of overexpressed Arl13b. While the motility of Arl13b overexpressing excessively long motile cilia was obviously disrupted, surprisingly, the abnormally long immotile primary cilia seemed to retain their signaling capacity. arl13b is induced by FoxJ1 and Rfx, and these ciliogenic transcription factors are unable to promote ciliary length increase when Arl13b activity is inhibited. Conversely, overexpression of Arl13b was sufficient to restore ciliary length in zebrafish embryos deficient in FoxJ1 function. We show that Arl13b increases cilia length by inducing protrusion of the ciliary membrane, which is then followed by the extension of the axonemal microtubules. Using mutant versions of Arl13b, one of which has been shown to be causative of the ciliopathy Joubert syndrome, we establish that the GTPase activity of the protein is essential for ciliary membrane extension. Taken together, our findings identify Arl13b as an important effector of ciliary membrane biogenesis and ciliary length regulation, and provide insights into possible mechanisms of dysfunction of the protein in Joubert syndrome.

Mutations in CCDC11, which encodes a coiled-coil containing ciliary protein, causes situs inversus due to dysmotility of monocilia in the left-right organizer.

In vertebrates, establishment of left-right (LR) asymmetry is dependent on cilia-driven fluid flow within the LR organizer. Mutations in CCDC11 disrupt LR asymmetry in humans, but how the gene functions in LR patterning is presently unknown. We describe a patient with situs inversus totalis carrying homozygous loss-of-function mutations in CCDC11. We show that CCDC11 is an axonemal protein in respiratory cilia, but is largely dispensable for their structure and motility. To investigate the role of CCDC11 in LR development, we studied the zebrafish homolog of the gene. Like in human respiratory cilia, loss of Ccdc11 causes minor defects in the motility of zebrafish kidney cilia, although the protein localizes to their axonemes and base. By contrast, Ccdc11 localizes exclusively to the basal bodies of cilia within Kupffer's vesicle, the organ of laterality of teleost fishes, and within the spinal canal. Moreover, the rotational motion of the cilia in these tissues of ccdc11-deficient embryos was strongly impaired. Our findings demonstrate that CCDC11 has a conserved essential function in cilia of the vertebrate LR organizer. To the best of our knowledge, this is the first ciliary component, which has a differential localization and function in different kinds of motile cilia.

Evolutionarily ancient association of the FoxJ1 transcription factor with the motile ciliogenic program.

It is generally believed that the last eukaryotic common ancestor (LECA) was a unicellular organism with motile cilia. In the vertebrates, the winged-helix transcription factor FoxJ1 functions as the master regulator of motile cilia biogenesis. Despite the antiquity of cilia, their highly conserved structure, and their mechanism of motility, the evolution of the transcriptional program controlling ciliogenesis has remained incompletely understood. In particular, it is presently not known how the generation of motile cilia is programmed outside of the vertebrates, and whether and to what extent the FoxJ1-dependent regulation is conserved. We have performed a survey of numerous eukaryotic genomes and discovered that genes homologous to foxJ1 are restricted only to organisms belonging to the unikont lineage. Using a mis-expression assay, we then obtained evidence of a conserved ability of FoxJ1 proteins from a number of diverse phyletic groups to activate the expression of a host of motile ciliary genes in zebrafish embryos. Conversely, we found that inactivation of a foxJ1 gene in Schmidtea mediterranea, a platyhelminth (flatworm) that utilizes motile cilia for locomotion, led to a profound disruption in the differentiation of motile cilia. Together, all of these findings provide the first evolutionary perspective into the transcriptional control of motile ciliogenesis and allow us to propose a conserved FoxJ1-regulated mechanism for motile cilia biogenesis back to the origin of the metazoans.