Research ArticleDEVELOPMENTAL BIOLOGY

Primary cilium remodeling mediates a cell signaling switch in differentiating neurons

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Science Advances  20 May 2020:
Vol. 6, no. 21, eabb0601
DOI: 10.1126/sciadv.abb0601
  • Fig. 1 The primary cilium is dynamically disassembled and reassembled during neuronal differentiation.

    (A) Time-lapse sequence showing Arl13b+ particle retention following apical abscission (movie S1). White arrows indicate apical abscission, white arrowheads indicate the retained Arl13b+ particle, and yellow arrowheads indicate the abscised primary cilium. Dashed magenta lines indicate ventricular surface. Scale bar, 10 μm. Boxed regions are enlarged in right-hand panels. Primary cilium is highlighted by white dashed lines. Scale bar, 0.7 μm. (B) Time-lapse sequence of a cell undergoing primary cilium reassembly during apical process retraction (movie S2). Cyan dashed lines demarcate cell body of differentiating neuron, and white arrowheads indicate the reassembling primary cilium. Boxed regions are enlarged in right-hand panels. Scale bars, 5 and 0.7 μm (enlarged region). (C) Cell undergoing axonogenesis transfected with Arl13b-GFP to label the ciliary membrane (green), mKate2-GPI to label cell membrane (red), and Neurog2 (Ngn2-GFP, green) (movie S3). Scale bars, 5 μm. (D) Representation of differentiating neurons at different stages of apical process retraction and early stages of axonogenesis. (E) Immunostaining to detect Tuj1+ cells (green) and endogenous Arl13b (red) at different stages of apical process retraction. Scale bars, 5 and 0.7 μm (enlarged region). (F) Quantification of absolute length of primary cilia (μm) from proximal-distal stages of apical process retraction in fixed tissue [mean ± SD, **P < 0.01 and ***P < 0.001, ordinary one-way analysis of variance (ANOVA) and Tukey’s post hoc test used for statistical analyses]. (G) Immunostaining to detect endogenous Arl13b and polyglutamylated tubulin (PolyGluTub) at different stages of apical process retraction. (H) Quantification of absolute length of the PolyGlu-labeled axoneme from proximal-distal stages of apical abscission in fixed tissue (mean ± SD, **P < 0.01 and ***P < 0.001, ordinary one-way ANOVA and Tukey’s post hoc test used for statistical analyses). ns, not significant.

  • Fig. 2 Intraflagellar trafficking is inactive in the Arl13+ particle and reactivated during apical process retraction.

    (A) Immunostaining to detect endogenous IFT88 (red), Arl13b (blue), and Tuj-1 (green). Cyan dashed lines demarcate differentiating cells, and boxed regions are enlarged in right-hand panels followed by a cartoon of representative IFT88 localization (BB, basal body). Primary cilium is highlighted by white dashed lines. Top right panels show primary cilium in cell body of Tuj1+ cell extending axon in the basal region of the spinal cord. Bottom right panel shows quantification of percentage of cells with detectable IFT88 in ciliary tip (present) at different stages of apical process retraction and axonogenesis. Scale bars, 5 and 0.7 μm (enlarged region). (B) Immunofluorescence and Airyscan enhanced-resolution microscopy of misexpressed PACT-TagRFP (red) and endogenous IFT88 (white). (C) Airyscan 3D reconstruction of primary cilia (red) at different stages of apical process retraction (stages are summarized in the diagram on the left; movies S4 to S7). Cyan dashed lines demarcate the tip of apical process, red dashed lines demarcate the centrosome, and white dashed lines demarcate the remodeled cilium. Scale bar, 1 μm. (D) Time-lapse sequence of differentiating neuron misexpressing IFT88-mNeonGreen undergoing apical process retraction (movie S8) and axonogenesis (movie S9). Cyan dashed lines outline differentiating cell. Enlarged regions indicate representative still images from time-lapse sequences. Blue arrows indicate IFT88 in reassembled primary cilium. Scale bars, 5 μm (process retraction), 10 μm (axonogenesis), and 0.7 μm (enlarged regions).

  • Fig. 3 The remodeled primary cilium is essential for establishment and subsequent maintenance of the nascent axon.

    (A) Immunostaining to detect Arl13b (white) in cells subjected and not subjected to green light irradiation. Boxed regions are enlarged in right-hand panels. Scale bars, 5 and 0.7 μm (enlarged region). IF, immunofluorescence. (B) PACT–YFP (yellow fluorescent protein) and Arl13b-SuperNova were misexpressed to label the centrosome in CALI experiments (movie S10). GFP fluorescence was compared before and after green light stimulation. Arrowhead indicates the centrosome. Scale bar, 0.7 μm. (C) Cell attached to the apical surface misexpressing Arl13b-SuperNova and EB3-mNeonGreen to label microtubule plus-ends (movie S11). Comets were tracked before and after CALI. Boxed regions are enlarged in bottom panels (before and after CALI) showing displacement of individual EB3-GFP comets. Scale bar, 5 μm. (D) Cell undergoing axonogenesis misexpressing Arl13b-SuperNova and EB3-mNeonGreen (movie S12). Comets were tracked before and after CALI. Boxed regions are enlarged in bottom panels (before and after CALI) showing displacement of individual EB3-GFP comets. (E) Time-lapse sequence of a cell undergoing apical process retraction following CALI-mediated disruption of the retained Arl13b+ particle (movie S13). Cyan dashed lines demarcate the differentiating cell, and the inset shows a zoomed-in view of the retained Arl13b+ particle highlighted by white dashed lines. Magenta arrowhead indicates the tip of the retracting process. Scale bar, 10 μm. (F) Time-lapse sequence of CALI-mediated disruption of primary cilium in cell undergoing axonogenesis and subsequent cell behavior. The inset shows a zoomed-in view of the primary cilium highlighted by white dashed lines. Yellow arrows indicate axon collapse (movie S14). Scale bar, 10 μm.

  • Fig. 4 Primary cilium remodeling corresponds with a switch to noncanonical Shh signaling.

    (A) Time-lapse sequence of a cell misexpressing Smo-GFP undergoing apical abscission (movie S22). Insets show an enlarged view of the primary cilium demarcated by white dashed lines. Scale bars, 5 and 0.7 μm (enlarged region). (B) Immunostaining to label endogenous Smo (white), Arl13b (red), and Tuj-1 (green). Bottom right corner shows quantification of Smo accumulation in primary cilia at different stages of apical process retraction (bottom right corner). Cyan dashed lines demarcate the differentiating cell, and the inset shows an enlarged view of the primary cilia demarcated by white dashed lines. Scale bars, 5 and 0.7 μm (enlarged region). (C) Time-lapse sequence of cells expressing the reporter of Gli activity GBS-GFP undergoing apical abscission (movie S23) and mitosis (movie S24). GFP fluorescence was normalized to the point previous to apical process retraction (C′) and the point of initiation of mitosis (C″). Scale bar, 5 μm. Arrows in C′ and C″ correspond to initiation of apical abscission and initiation of mitosis, respectively. Scale bar, 5 μm. (D) Immunostaining to label endogenous GPR161 (green), Arl13b (red), and Tuj1 (white). Right-hand panel shows quantification of GPR161 accumulation in primary cilia at different stages of apical process retraction. Cyan dashed lines demarcate the differentiating cell, and the insets show an enlarged view of the primary cilia demarcated by white dashed lines. Scale bars, 10 and 0.7 μm (enlarged region).

  • Fig. 5 Disruption of noncanonical Shh signaling leads to axon collapse.

    (A) Time-lapse sequence of cell undergoing axonogenesis imaged in medium containing the Smo antagonist cyclopamine (movie S25) and control medium containing ethanol (movie S26). Scale bar, 10 μm. (B) Time-lapse sequence of cell undergoing axonogenesis imaged in medium containing the SFK inhibitor PP2 (movie S27) and control medium containing dimethyl sulfoxide (DMSO) (movie S28). Scale bar, 5 μm.

  • Fig. 6 Final model.

    Remodeling of the primary cilium during neuronal differentiation directs the transition from canonical to noncanonical Shh signaling.

Supplementary Materials

  • Supplementary Materials

    Primary cilium remodeling mediates a cell signaling switch in differentiating neurons

    Gabriela Toro-Tapia and Raman M. Das

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