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Shh-mediated centrosomal recruitment of PKA promotes symmetric proliferative neuroepithelial cell division

Nature Cell Biology volume 19, pages 493–503 (2017)Cite this article

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Abstract

Tight control of the balance between self-expanding symmetric and self-renewing asymmetric neural progenitor divisions is crucial to regulate the number of cells in the developing central nervous system. We recently demonstrated that Sonic hedgehog (Shh) signalling is required for the expansion of motor neuron progenitors by maintaining symmetric divisions. Here we show that activation of Shh/Gli signalling in dividing neuroepithelial cells controls the symmetric recruitment of PKA to the centrosomes that nucleate the mitotic spindle, maintaining symmetric proliferative divisions. Notably, Shh signalling upregulates the expression of pericentrin, which is required to dock PKA to the centrosomes, which in turn exerts a positive feedback onto Shh signalling. Thus, by controlling centrosomal protein assembly, we propose that Shh signalling overcomes the intrinsic asymmetry at the centrosome during neuroepithelial cell division, thereby promoting self-expanding symmetric divisions and the expansion of the progenitor pool.

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Figure 1: Strong Shh/Gli activity in symmetrically dividing MN progenitors.
Figure 2: PKA localizes to the centrosome in dividing neural progenitors.
Figure 3: PKA docking to centrosomes changes during NT development.
Figure 4: Activation of the Shh/Gli pathway correlates with symmetric centrosomal docking of PKA.
Figure 5: Activation of the Shh/Gli pathway correlates with symmetric inheritance of apical membrane domains.
Figure 6: Asymmetric centrosomal docking of PKA is correlated with neurogenic divisions.
Figure 7: Shh signalling regulates pericentrin expression.
Figure 8: Pericentrin-mediated PKA docking to the centrosomes is necessary for Shh/Gli activation and inhibition of neurogenesis.

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Acknowledgements

The authors are indebted to E. Rebollo for her invaluable technical assistance at the AFMU Facility (IBMB). For providing DNAs, we thank S. McKnight (University of Washington, USA), M. Uchikawa (Osaka University, Japan), M. Götz (Ludwig-Maximilians-University Munich, Germany), H. Lickert (GmbH, ISF, Neuherberg, Germany) and S. Pons (IBMB-CSIC). For providing antibodies, we also thank O. Rosnet (CRCM, Marseille, France), M. Bornens (Institut Curie, Paris, France) and S. Pons (IBMB-CSIC). The monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank, developed under the auspices of the NICHD and maintained by The University of Iowa, Department of Biological Sciences, Iowa City, Iowa 52242. The work in E.M.’s laboratory was supported by grants BFU2013-46477-P and BFU2014-55738-REDT.

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  1. Rene Escalona

    Present address: Present address: Departamento de Embriologia, Facultad de Medicina, Universidad Nacional Autónoma de México, Ciudad de Mexico CP 04510, Mexico.,

Authors and Affiliations

  1. Instituto de Biología Molecular de Barcelona, CSIC, ParcCientífic de Barcelona, C/Baldiri i Reixac 20, Barcelona 08028, Spain

    Murielle Saade, Elena Gonzalez-Gobartt, Rene Escalona, Susana Usieto & Elisa Martí

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Contributions

M.S. conceived and performed most experiments, analysed the data and discussed results. E.G.-G. contributed to image acquisition, image analysis and quantification, and statistics. R.E. performed the luciferase experiments. S.U. provided technical support to all experiments. E.M. conceived experiments, analysed the data, discussed results and wrote the manuscript.

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Correspondence to Elisa Martí.

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Integrated supplementary information

Supplementary Figure 1 In dividing neuroepithelial cells, the primary cilia was not completely disassembled prior to mitosis.

(a) Transient expression of CEP152-GFP in the chick NT after electroporation (HH14, 16 h post electroporation, hpe) reliably labels the two centrosomes in dividing neural progenitors. CEP152-GFP (green) formed pairs of dots at the spindle poles during mitosis that co-localize with a-Tubulin-GFP (green) and that are immunostained with anti-α-Tubulin (red). DAPI (blue) stains the chromosomes (scale bars, 5 μm). (b) α-Tubulin-GFP (green) electroporation labelled the mitotic spindle. Immunostaining with anti-FOP (FGFR1 Oncogene Partner, red) revealed the centrosome pairs lining the NT lumen, as well as the nucleating mitotic spindles (Scale bars 5 μm). (c) Gli3-HA (red) localizes to the cilium tip (pink arrows) and the nucleus. Acetylated tubulin (green) stain the cilium shaft (yellow arrow) (d) Scheme showing the DNAs and timing of the co-electroporation (hpe = hours post electroporation) (scale bars, are 10 μm and 2 μm respectively). (e) Quantification of the subcellular Arl13b localization types as percentage of total anaphase/telophase H2B-GFP+ mitoses, at two developmental stages, showing that as neurogenesis progresses, the Arl13b-labelled ciliary remnant can lose its attachment to the old mother centriole during mitosis (HH10, n = 30 mitoses; HH14 n = 30 mitoses, from three independent experiments). (f,g) Selected images showing non-centrosomal (f) and centrosomal (g) Arl13b localization; H2B-GFP (green) labels chromosomes, anti-FOP (blue) revealed the centrosome pairs, Arl13b-RFP (red) labels the ciliary remnant. Yellow arrow shows centrosome localization (FOP+) and purple arrow shows ciliary remnant localization (Arl13b+) (Scale bars 5 μm). Images are representative of three independent experiments.

Supplementary Figure 2 PKA localizes to the centrosomes in dividing neural progenitors throughout mitosis.

(a,b) Transient expression of Arl13b-RFP in the chick NT (HH14, 16 hpe) reliably labels the primary cilia: centrosomes labelled with anti-FOP (purple) line the NT lumen; Arl13b-RFP (red) labels the cilia at the NT lumen; anti-FLAG staining (green) labels PKA; DAPI (blue) labels the nuclei (scale bar, 10 μm in a; scale bar, 0,5 μm in b). (c) RII-PKA and Cα-PKA are always co-electroporated in order to inhibit the enzyme’s kinase activity. Co-electroporation at low concentrations of RII-PKA + Cα-PKA was used to study the subcellular localization, and they do not activate Shh transcriptional responses, as assessed by the Gli-BS-Luc reporter activity. Both dnPKA and SmoM2 are assessed as activators of the pathway, and PtcΔ Loop2 is studied as an inhibitor of the pathway. Quantification of the Luc/Renilla activity of the Gli-BS-Luc reporter 24 hpe of the DNAs indicated (plots show the mean ± s.e.m., n = 8 embryos per condition; three independent experiments; one-way ANOVA; ∗P < 0.05, ∗∗∗P < 0.001) (scale bars, 5 μm). (d–i) PKA localizes to the centrosomes at different mitotic phases. RII-PKA + Cα-PKA-FLAG, revealed Cα-PKA by anti-FLAG staining (green) at centrosomes labelled with anti-FOP (red), from prophase to cytokinesis. DAPI (blue) labels the chromosomes. (j,k) Endogenous RII-PKA (green) and Cα-PKA (red) subunits symmetrically localize to centrosomes labelled with anti-FOP (purple), during mitosis. DAPI (blue) labels the chromosomes. (l,m) Endogenous RII-PKA (green) and Cα-PKA (red) subunits asymmetrically localize to centrosomes labelled with anti-FOP (purple), during mitosis. DAPI (blue) labels the chromosomes. (n,o) Endogenous RII-PKA (green) and Cα-PKA (red) subunits co-localize, either symmetrically (n) or asymmetrically (o) to centrosomes labelled with anti-FOP (purple), during mitosis. DAPI (blue) labels the chromosomes (scale bars, 5 μm). Images are representative of three independent experiments.

Supplementary Figure 3 PKA dissociates from centrosomes at the onset of neurogenesis,

(a) Scheme showing the DNAs electroporated and the timing of electroporation. (b) Selected images showing the symmetric centrosomal docking of PKA at the base of the cilium in Gli-BS-RFP+ (red) sister cells. Cα-PKA + RII-PKA-FLAG electroporation revealed Ca-PKA by anti-FLAG staining (green) and the centrosomes were labelled with anti-FOP (purple). (c,d) Selected images showing Gli-BS-RFP cells exiting the ventricular zone in which PKA is distributed in the cytosol but not predominantly associated to the FOP stained centrosome (yellow arrow). (e) Scheme showing the DNAs electroporated, the timing of electroporation, and the area (ROI) selected for fluorescence intensity measurement. (f) Quantification of centrosomal RII-PKA and Ca-PKA in Tis21-, plots show the mean ± s.e.m., Mann–Whitney U test, ∗P < 0.05,∗∗P < 0.01, of cumulative fluoresce intensity in both centrosomes (RII-PKA-FLAG mean = 41 ± 9, n = 19 mitoses; Ca-PKA mean = 41 ± 7, n = 20 mitoses) and Tis21 + (RII-PKA-FLAG mean = 20 ± 3,n = 22 mitoses; Ca-PKA mean = 17 ± 2, n = 26 mitoses; from three independent experiments). (g) Scheme showing electroporated cDNAs, and the timing of electroporation. (h) Selected images showing asymmetric centrosomal docking of PKA in pTis21+ sister cells (red). Cα-PKA + RII-PKA-FLAG electroporation revealed Cα-PKA by anti-FLAG staining (green), showing the asymmetric docking of PKA at the centrosomes lining the NT lumen (yellow arrows), labelled with anti-FOP (purple) (scale bar, 0,5 μm). (i) Selected images showing pTis21+ cells exiting the ventricular zone where PKA is distributed in the cytosol and not associated to the FOP (purple) stained centrosomes (yellow arrow). (j) High magnification of the apical area in I, showing the centrosomal duplication (two yellow arrows) in the daughter cell that remains as a progenitor, in which PKA remains docked to the apical centrosomes (scale bars, 10 μm). (k) Scheme showing the area (ROI) selected for fluorescence intensity measurement. (l) Quantification of the endogenous centrosomal RII-PKA and endogenous ninein, plots show the mean ± s.e.m., Mann–Whitney U test, ∗∗P < 0.01, ∗∗∗P < 0.001 of cumulative fluoresce intensity in mother (high ninein) centrosomes (RII-PKA mean = 20, 6 ± 3,5 ninein mean = 9, 2 ± 1,6) and in dauther (low ninein) centrosomes (RII-PKA mean = 5, 8 ± 1, 3 ninein mean = 1, 8 ± 0, 8 n = 11 mitoses) Divisions were analysed from 6 independent embryos in one experiment. Images are representative of three independent experiments.

Supplementary Figure 4 Neurogenesis correlates with asymmetric inheritance of apical membrane domains.

(a) Scheme showing the split of AJs by the cleavage plane at anaphase, the cleavage plane being deduced by a line bisecting the two sets of condensed chromatin (blue plates). When the rectangles were oriented parallel to each other, the cleavage plane was positioned half way in between the rectangles (d1 = d2). When the rectangles were oriented at an angle to each other, the cleavage plane was positioned such that this angle was halved (a1 = a2), predicting the type of division made by distributing the N-cadherin hole (red outline) between the two daughter cells, and by partitioning the apical aPKC domain (green). (b) Quantification of the Rfi between inherited αPKC apical domains in mitotic cells at two developmental stages, where the lines and error bars correspond to the median ± s.e.m.; three independent experiments; Mann–Whitney U test; ∗∗∗P < 0.001. The partitioning of αPKC was largely symmetric at 54 hpf; n = 32 mitosis), while it was asymmetric at the neurogenic phase 70 hpf (n = 42 mitosis). (c) Example of a symmetric mitosis in which the cleavage plane is deduced by drawing perpendicular lines (dashed line) bisecting the two sets of condensed chromatin (outlined). DAPI (blue) labels the chromosomes, N-cadherin is labelled in red, αPKC in green. (d) Example of an asymmetric mitosis (scale bars, 5 μm). (e) Scheme showing the DNAs electroporated and the timing of electroporation. (f) Example of two cells in anaphase and telophase in which the cleavage plane is deduced by drawing perpendicular lines (dashed line) bisecting the two sets of condensed chromatin (outlined). Symmetric partitioning of αPKC is correlated with Gli-BS activation (RFP+). GFP represents the control electroporation, FOP (purple) labels the centrosomes, DAPI (blue) labels the chromosomes (yellow arrow points to the N-cadherin hole). Black and white panel shows the isolation of αPKC domain for quantification. (g) Example of two cells in anaphase and telophase, in which asymmetric partitioning of αPKC is correlated with Gli-BS inactivation (RFP−, yellow arrow points to the N-cadherin hole) (scale bars, 5 μm). (h) Scheme showing the DNAs electroporated and the timing of electroporation. (i) Example of two cells in telophase, in which asymmetric partitioning of αPKC is correlated with asymmetric centrosomal docking of RII-PKA. FOP (magenta) labels the centrosomes (magenta arrows point to centrosomes). RFP (red) labels the chromosomes (yellow arrow points to the N-cadherin hole)(Scale bars 5 μm). Images are representative of three independent experiments.

Supplementary Figure 5 Pericentrin mediates PKA docking to the centrosomes.

(a) Semi-quantitative PCR analysis of the AKAP9 transcripts expressed in control versus SmoM2-EP cells (plot shows the mean ± s.e.m., 25.000 cells from n = 8 independent embryos, three independent experiments, Mann–Whitney U test; NS). (b) Scheme showing the conservation of the PCNT (pericentrin) sequence, highlighting the RII-PKA-binding domain (blue) and the PCNT-AKAP9 Centrosomal Targeting (PACT) domain (purple). (c) Multiple sequence alignment of the RII-PKA binding domain of PCNT using Multaling version 5.4.1: red highlights the conserved leucines (L) critical for RII-PKA binding. (d) Multiple sequence alignment highlighting the conserved PACT binding domain in PCNT. (e) Selected images of RIIΔ2-6-PKA electroporation at mitotic phases: CEP152 (red) labels centrosomes (yellow arrows); DAPI (blue) labels chromosomes. Mutant RII-PKA (green) does not associate with the centrosomes at any phase of mitosis (scale bars, 5 μm Images are representative of three independent experiments.

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3D reconstruction of symmetric centrosomal docking of PKA in a Gli-BS-RFP+ mitoses.

Gli-BS-RFP+ mitosis (red) show symmetric RII-PKA/Cα-PKA-FLAG, revealed Cα-PKA by anti-FLAG staining (green) in centrosomes labelled with anti-FOP (purple). DAPI (blue) labels the chromosomes and yellow arrows point to PKA. (MP4 1798 kb)

3D reconstruction of asymmetric centrosomal docking of PKA in a pTis21-RFP+ mitoses.

pTis21-RFP+ mitoses (red) show asymmetric RII-PKA/Cα-PKA-FLAG, revealed Cα-PKA by anti-FLAG staining (green) in centrosomes labelled with anti-FOP (purple). DAPI (blue) labels the chromosomes and yellow arrows point to PKA. (MP4 4395 kb)

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Saade, M., Gonzalez-Gobartt, E., Escalona, R. et al. Shh-mediated centrosomal recruitment of PKA promotes symmetric proliferative neuroepithelial cell division. Nat Cell Biol 19, 493–503 (2017). https://doi.org/10.1038/ncb3512

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