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During the development of the cerebral cortex, neurons are generated directly from radial glial cells or indirectly via basal progenitors. The balance between these division modes determines the number and types of neurons formed in the cortex thereby affecting cortical functioning. Here, we investigate the role of primary cilia in this process. We show that a mutation in the ciliary geneInpp5eleads to a transient increase in direct neurogenesis and subsequently to an overproduction of layer V neurons in newborn mice. Loss ofInpp5ealso affects ciliary structure coinciding with increased Akt and mTOR signalling and reduced Gli3 repressor levels. Genetically re-storing Gli3 repressor rescues the decreased indirect neurogenesis inInpp5emutants. Overall, our analyses reveal how primary cilia determine neuronal subtype composition of the cortex by controlling direct vs indirect neurogenesis. These findings have implications for understanding cortical malformations in ciliopathies withINPP5Emutations.

A range of severe human diseases called ciliopathies are caused by the dysfunction of primary cilia. Primary cilia are cytoplasmic protrusions consisting of the basal body (BB), the axoneme and the transition zone (TZ). The BB is a modified mother centriole from which the axoneme, the microtubule-based ciliary scaffold, is formed. At the proximal end of the axoneme, the TZ functions as the ciliary gate governing ciliary protein entry and exit. Since ciliopathies often develop due to mutations in genes encoding proteins that localise to the TZ, the understanding of the mechanisms underlying TZ function is of eminent importance. Here, we show that the ciliopathy protein Rpgrip1l governs ciliary gating by ensuring the proper amount of Cep290 at the vertebrate TZ. Further, we identified the flavonoid eupatilin as a potential agent to tackle ciliopathies caused by mutations in RPGRIP1L as it rescues ciliary gating in the absence of Rpgrip1l.

SUMMARYCilia-driven movements of the cerebrospinal fluid (CSF) are involved in zebrafish axis straightness, both in embryos and juveniles [1, 2]. In embryos, axis straightness requires cilia-dependent assembly of the Reissner fiber (RF), a SCO-spondin polymer running down the brain and spinal cord CSF-filled cavities [3]. Reduced expression levels of the urp1 and urp2 genes encoding neuropeptides of the Urotensin II family in CSF-contacting neurons (CSF-cNs) also underlie embryonic ventral curvature of several cilia motility mutants [4]. Moreover, mutants for scospondin and uts2r3 (a Urotensin II peptide family receptor gene) develop scoliosis at juvenile stages [3, 4]. However, whether RF maintenance and URP signaling are perturbed in juvenile scoliotic ciliary mutants and how these perturbations are linked to scoliosis is unknown. Here we produced mutants in the zebrafish ortholog of the human RPGRIP1L ciliopathy gene encoding a transition zone protein [5–7]. rpgrip1l-/- zebrafish had normal embryogenesis and developed 3D spine torsions in juveniles. Cilia lining the CNS cavities were normal in rpgrip1l-/- embryos but sparse and malformed in juveniles and adults. Hindbrain ventricle dilations were present at scoliosis onset, suggesting defects in CSF flow. Immunostaining showed a secondary loss of RF correlating with juvenile scoliosis. Surprisingly, transcriptome analysis of rgprip1l mutants at scoliosis onset uncovered increased levels of urp1 and urp2 expression. Overexpressing urp2 in foxj1-expressing cells triggered scoliosis in rpgrip1l heterozygotes. Thus, our results demonstrate that increased URP signaling drives scoliosis onset in a ciliopathy mutant. We propose that imbalanced levels of URP neuropeptides in CSF-cNs may be an initial trigger of scoliosis.

Ciliogenesis is a general process in eukaryotic cells and its different steps begin to be well characterised. However, the molecular mechanisms leading to decilation or ciliary shedding are still poorly understood. This process, observed from unicellular organisms such asChlamydomonasorParameciumto multiciliated cells from trachea or fallopian tube of vertebrates, seems to be a general process since recent observations demonstrates its requirement during the cell cycle or neurogenesis. Interestingly, in all cellular models, ciliary shedding occurs distal to the transition zone, essentially known to act as a diffusion barrier between the intracellular space and the cilium, suggesting conserved molecular mechanisms.To determine if MKS and NPHP modules, known to cooperate to establish transition zone formation and function, could control ciliary shedding, we studied inParameciumthe function of TMEM216/MKS2 and TMEM107 (two members of the MKS module), NPHP4 (one member of the NPHP module), CEP290/NPHP6 and RPGRIP1L/MKS5. We show that all these proteins are recruited to the TZ as soon as growing cilia are detected and localise with a 9-fold symmetry at the level of the axonemal plate. Interestingly, we demonstrate that the depletion of the two MKS module proteins induces spontaneous cilia shedding, while the depletion of either NPHP4, CEP290 or RPGRIP1L inhibits the process. Our results constitute the first evidence for a role of conserved TZ proteins in deciliation and open new directions for understanding motile cilia physiology.