Multiciliate cells function prominently in the respiratory system, brain ependyma, and female reproductive tract to produce vigorous fluid flow along epithelial surfaces. These specialized cells form during development when epithelial progenitors undergo an unusual form of ciliogenesis, in which they assemble and project hundreds of motile cilia. Notch inhibits multiciliate cell formation in diverse epithelia, but how progenitors overcome lateral inhibition and initiate multiciliate cell differentiation is unknown. Here we identify a coiled-coil protein, termed multicilin, which is Notch regulated and highly expressed in developing epithelia where multiciliate cells form. Inhibiting multicilin function specifically blocks multiciliate cell formation in the Xenopus skin and kidney, while ectopic expression induces the differentiation of multiciliate cells in ectopic locations. Multicilin localizes to the nucleus, where it directly activates the expression of genes required for multiciliate cell formation, including FoxJ1 and genes mediating centriole assembly. Multicilin is also necessary and sufficient to promote multiciliate cell differentiation in mouse airway epithelial cultures. These findings suggest that multicilin initiates multiciliate cell differentiation in diverse tissues, by coordinately promoting the transcriptional changes required for motile ciliogenesis and centriole assembly.

SummaryMotile cilia produce large-scale fluid flows critical for development and physiology. Defects in ciliary motility cause a range of disease symptoms including bronchiectasis, hydrocephalus, and situs inversus. However, it is not enough for cilia to be motile and generate a flow -the flow must be driven in the proper direction. Generation of properly directed coherent flow requires that the cilia are properly oriented relative to tissue axes. Genetic, molecular, and ultrastructural studies have begun to suggest pathways linking cilia orientation to planar cell polarity and other long-range positional cues, and also suggest that cilia-driven flow can itself play a causal role in orienting the cilia that create it. Errors in cilia orientation have been observed in human ciliary disease patients, suggesting that orientation defects may constitute a novel class of ciliopathies with a distinct etiology at the cell biological level.

Motor output varies along the rostro-caudal axis of the tetrapod spinal cord. At limb levels, ∼60 motor pools control the alternation of flexor and extensor muscles about each joint, whereas at thoracic levels as few as 10 motor pools supply muscle groups that support posture, inspiration, and expiration. Whether such differences in motor neuron identity and muscle number are associated with segmental distinctions in interneuron diversity has not been resolved. We show that select combinations of nineteen transcription factors that specify lumbar V1 inhibitory interneurons generate subpopulations enriched at limb and thoracic levels. Specification of limb and thoracic V1 interneurons involves the Hox gene Hoxc9 independently of motor neurons. Thus, early Hox patterning of the spinal cord determines the identity of V1 interneurons and motor neurons. These studies reveal a developmental program of V1 interneuron diversity, providing insight into the organization of inhibitory interneurons associated with differential motor output.

V1 interneurons are inhibitory neurons that play an essential role in vertebrate locomotion. The molecular mechanisms underlying their genesis remain, however, largely undefined. Here, we show that the transcription factor Prdm12 is selectively expressed in p1 progenitors of the hindbrain and spinal cord in the frog embryo, and that a similar restricted expression profile is observed in the nerve cord of other vertebrates as well as of the cephalochordate amphioxus. Using frog, chick and mice, we analyzed the regulation of Prdm12 and found that its expression in the caudal neural tube is dependent on retinoic acid and Pax6, and that it is restricted to p1 progenitors, due to the repressive action of Dbx1 and Nkx6-1/2 expressed in the adjacent p0 and p2 domains. Functional studies in the frog, including genomewide identification of its targets by RNA-seq and ChIP-Seq, reveal that vertebrate Prdm12 proteins act as a general determinant of V1 cell fate, at least in part, by directly repressing Dbx1 and Nkx6 genes. This probably occurs by recruiting the methyltransferase G9a, an activity that is not displayed by the amphioxus Prdm12 protein. Together, these findings indicate that Prdm12 promotes V1 interneurons through cross-repressive interactions with Dbx1 and Nkx6 genes, and suggest that this function might have only been acquired after the split of the vertebrate and cephalochordate lineages.

Upon cell cycle exit, centriole-to-basal body transition facilitates cilia formation. The centriolar protein Cp110 is a regulator of this process and cilia inhibitor, but its positive roles in ciliogenesis remain poorly understood. Using Xenopus we show that Cp110 inhibits cilia formation at high levels, while optimal levels promote ciliogenesis. Cp110 localizes to cilia-forming basal bodies and rootlets, and is required for ciliary adhesion complexes that facilitate Actin interactions. The opposing roles of Cp110 in ciliation are generated in part by coiled-coil domains that mediate preferential binding to centrioles over rootlets. Because of its dual role in ciliogenesis, Cp110 levels must be precisely controlled. In multiciliated cells, this is achieved by both transcriptional and posttranscriptional regulation through ciliary transcription factors and microRNAs, which activate and repress cp110 to produce optimal Cp110 levels during ciliogenesis. Our data provide novel insights into how Cp110 and its regulation contribute to development and cell function.