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Arl13b-regulated cilia activities are essential for polarized radial glial scaffold formation.

ABSTRACT: The construction of cerebral cortex begins with the formation of radial glia. Once formed, polarized radial glial cells divide either symmetrically or asymmetrically to balance appropriate production of progenitor cells and neurons. Following birth, neurons use the processes of radial glia as scaffolding for oriented migration. Radial glia therefore provide an instructive structural matrix to coordinate the generation and placement of distinct groups of cortical neurons in the developing cerebral cortex. We found that Arl13b, a cilia-enriched small GTPase that is mutated in Joubert syndrome, was critical for the initial formation of the polarized radial progenitor scaffold. Using developmental stage-specific deletion of Arl13b in mouse cortical progenitors, we found that early neuroepithelial deletion of ciliary Arl13b led to a reversal of the apical-basal polarity of radial progenitors and aberrant neuronal placement. Arl13b modulated ciliary signaling necessary for radial glial polarity. Our findings indicate that Arl13b signaling in primary cilia is crucial for the initial formation of a polarized radial glial scaffold and suggest that disruption of this process may contribute to aberrant neurodevelopment and brain abnormalities in Joubert syndrome-related ciliopathies.

SUBMITTER: Higginbotham H

PROVIDER: S-EPMC3866024 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Arl13b-regulated cilia activities are essential for polarized radial glial scaffold formation.

Higginbotham Holden H Guo Jiami J Yokota Yukako Y Umberger Nicole L NL Su Chen-Ying CY Li Jingjun J Verma Nisha N Hirt Joshua J Ghukasyan Vladimir V Caspary Tamara T Anton E S ES

Nature neuroscience 20130630 8

The construction of cerebral cortex begins with the formation of radial glia. Once formed, polarized radial glial cells divide either symmetrically or asymmetrically to balance appropriate production of progenitor cells and neurons. Following birth, neurons use the processes of radial glia as scaffolding for oriented migration. Radial glia therefore provide an instructive structural matrix to coordinate the generation and placement of distinct groups of cortical neurons in the developing cerebra ...[more]

PMID: 23817546

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Project description:BACKGROUND:The primary cilium is an extension of the cell membrane that encloses a microtubule-based axoneme. Primary cilia are essential for transmission of environmental cues that determine cell fate. Disruption of primary cilia function is the molecular basis of numerous developmental disorders. Despite their biological importance, the mechanisms governing their assembly and disassembly are just beginning to be understood. Cilia growth and disassembly are essential events when cells exit and reenter into the cell cycle. The kinases never in mitosis-kinase 2 (Nek2) and Aurora A (AurA) act to depolymerize cilia when cells reenter the cell cycle from G0. RESULTS:Coexpression of either kinase with its kinase dead companion [AurA with kinase dead Nek2 (Nek2 KD) or Nek2 with kinase dead AurA (AurA KD)] had different effects on cilia depending on whether cilia are growing or shortening. AurA and Nek2 are individually able to shorten cilia when cilia are growing but both are required when cilia are being absorbed. The depolymerizing activity of each kinase is increased when coexpressed with the kinase dead version of the other kinase but only when cilia are assembling. Additionally, the two kinases act additively when cilia are assembling but not disassembling. Inhibition of AurA increases cilia number while inhibition of Nek2 significantly stimulates cilia length. The complex functional relationship between the two kinases reflects their physical interaction. Further, we identify a role for a PP1 binding protein, PPP1R42, in inhibiting Nek2 and increasing ciliation of ARPE-19 cells. CONCLUSION:We have uncovered a novel functional interaction between Nek2 and AurA that is dependent on the growth state of cilia. This differential interdependence reflects opposing regulation when cilia are growing or shortening. In addition to interaction between the kinases to regulate ciliation, the PP1 binding protein PPP1R42 directly inhibits Nek2 independent of PP1 indicating another level of regulation of this kinase. In summary, we demonstrate a complex interplay between Nek2 and AurA kinases in regulation of ciliation in ARPE-19 cells.

Project description:PurposeMutations in the gene ARL13B cause the classical form of Joubert syndrome, an autosomal recessive ciliopathy with variable degrees of retinal degeneration. As second-site modifier alleles can contribute to retinal pathology in ciliopathies, animal models provide a unique platform to test how genetic interactions modulate specific phenotypes. In this study, we analyzed the zebrafish arl13b mutant for retinal degeneration and for epistatic relationships with the planar cell polarity protein (PCP) component vangl2.MethodsPhotoreceptor and cilia structure was examined by light and electron microscopy. Immunohistochemistry was performed to examine ciliary markers. Genetic interactions were tested by pairwise crosses of heterozygous animals. Genetic mosaic animals were generated by blastula transplantation and analyzed by fluorescence microscopy.ResultsAt 5 days after fertilization, photoreceptor outer segments were shorter in zebrafish arl13b-/- mutants compared to wild-type larvae, no overt signs of retinal degeneration were observed by light or electron microscopy. Starting at 14 days after fertilization (dpf) and continuing through 30 dpf, cells lacking Arl13b died following transplantation into wild-type host animals. Photoreceptors of arl13b-/-;vangl2-/- mutants were more compromised than the photoreceptors of single mutants. Finally, when grown within a wild-type retina, the vangl2-/- mutant cone photoreceptors displayed normal basal body positioning.ConclusionsWe show that arl13b-/- mutants have shortened cilia and photoreceptor outer segments and exhibit a slow, progressive photoreceptor degeneration that occurs over weeks. The data suggest that loss of Arl13b leads to slow photoreceptor degeneration, but can be exacerbated by the loss of vangl2. Importantly, the data show that Arl13b can genetically and physically interact with Vangl2 and this association is important for normal photoreceptor structure. The loss of vangl2, however, does not affect basal body positioning.

Dataset Information

Arl13b-regulated cilia activities are essential for polarized radial glial scaffold formation.

Publications

Arl13b-regulated cilia activities are essential for polarized radial glial scaffold formation.

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