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Cortical neurogenesis in the absence of centrioles

Nature Neuroscience volume 17, pages 1528–1535 (2014)Cite this article

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Abstract

Neuronal production in the mammalian cortex depends on extensive mitoses of radial glial progenitors (RGPs) residing in the ventricular zone (VZ). We examined the function of centrioles in RGPs during cortical neurogenesis in mice by conditional removal of SAS-4, a protein that is required for centriole biogenesis. SAS-4 deletion led to a progressive loss of centrioles, accompanied by RGP detachment from the VZ. Delocalized RGPs did not become outer subventricular zone RGPs (oRGs). Although they remained proliferative, ectopic RGPs, as well as those in the VZ, with a centrosomal deficit exhibited prolonged mitosis, p53 upregulation and apoptosis, resulting in neuronal loss and microcephaly. Simultaneous removal of p53 fully rescued RGP death and microcephaly, but not RGP delocalization and randomized mitotic spindle orientation. Our findings define the functions of centrioles in anchoring RGPs in the VZ and ensuring their efficient mitoses, and reveal the robust adaptability of RGPs in the developing cortex.

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Figure 1: Sas-4 deletion causes microcephaly with widespread p53 upregulation and apoptosis.
Figure 2: Simultaneous removal of p53 rescues microcephaly caused by SAS-4 ablation.
Figure 3: Sas-4 deletion leads to RGP delocalization and loss.
Figure 4: Displaced RGPs lack centrioles/centrosomes and are not oRGs.
Figure 5: Displaced RGPs remain proliferative.
Figure 6: Centrosome loss causes mitotic delay.
Figure 7: VZ niche and cleavage plane orientation are not essential for consecutive rounds of RGP division and cortical neurogenesis.

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Acknowledgements

We thank B.M.-F. Tsou (Memorial Sloan Kettering Cancer Center) for reagents and advice, P. Gönczy (Institut Suisse de Recherches Experimentales sur le Cancer) for reagents, D. Solit (Memorial Sloan Kettering Cancer Center) for p53loxP/loxP mice, and V. Boyko and Y. Romin at the Molecular Cytology Core Facility for their technical support. We thank the Anderson and Shi laboratory members for their input and comments on the manuscript. This work was supported by grants from the US National Institutes of Health (R01DA024681 and R01NS085004 to S.-H.S., and R01NS044385 to K.V.A.). H.B. was supported by the National Kidney Foundation and a Ruth L. Kirschstein National Research Service Award from the US National Institutes of Health.

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Author notes

  1. Ryan Insolera and Hisham Bazzi: These authors contributed equally to this work.

Authors and Affiliations

  1. Developmental Biology Program, Memorial Sloan Kettering Cancer Center, New York, New York, USA

    Ryan Insolera, Hisham Bazzi, Wei Shao, Kathryn V Anderson & Song-Hai Shi

  2. Graduate Program in Neuroscience, Weill Cornell Medical College, New York, New York, USA

    Ryan Insolera & Song-Hai Shi

  3. Graduate Program in Biochemistry, Cell and Molecular Biology Allied Program, Weill Cornell Medical College, New York, New York, USA

    Wei Shao & Song-Hai Shi

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Contributions

R.I., H.B., K.V.A. and S.-H.S. conceived the project and designed the experiments. R.I. and W.S. analyzed the brain phenotype and H.B. generated the mutant animals. All of the authors discussed the results and contributed to the preparation of the manuscript.

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Correspondence to Kathryn V Anderson or Song-Hai Shi.

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Integrated supplementary information

Supplementary Figure 1 Efficient recombination in the developing cortex driven by Nestin-Cre and Emx1-Cre.

Representative images of E12.5 brains of Nestin-Cre (a) and Emx1-Cre (b) mice crossed with Ai9/tdTomato (red) mice stained with DAPI (blue). High magnification images of the cortex are shown at the bottom. Note the broad expression of tdTomato in the developing cortex, indicating efficient recombination driven by these two Cre lines. Scale bars: 100 μm (top) and 50 μm (bottom).

Supplementary Figure 2 Removal of p53 suppresses apoptosis and microcephaly in the Sas-4−/− cortex.

(a) Western blot analysis of SAS-4 expression in the WT (left, Sas-4+/–; right, Sas-4+/– p53−/−) and conditional mutant (left, Sas-4−/−; right, Sas-4−/− p53−/−) brains at E13.5 (left) and E15.5 (right). Note that SAS-4 is largely undetectable in the conditional mutant brain. (b) Representative whole mount images of E15.5 WT, Sas-4−/−, and Sas-4−/− p53−/− brains. Scale bar: 0.5 mm. (c) Representative images of E15.5 WT, Sas-4−/−, and Sas-4−/− p53−/− E15.5 cortices stained for CASP3 (green) and DAPI (blue). Note that the widespread apoptosis in the Sas-4−/− cortex is not observed in the Sas-4−/− p53−/− cortex. Scale bar: 50 μm.

Supplementary Figure 3 Removal of p53 does not impair cortical development.

(a) Representative whole mount images showing no obvious difference between size of WT and p53−/− brains at E15.5. Scale bar: 0.5 mm. (b, d, f) Representative images of E15.5 WT and p53−/− cortices stained for PAX6 (green, b), TBR2 (red, d), or PHH3 (red, f) and DAPI (blue). Note that there is no obvious depletion or delocalization of RGPs or IPs or change in mitosis in the p53−/− cortex compared to the WT control. Scale bars: 50 μm. (c, e, g) Quantification of the number of cells that are positive for PAX6 (c), TBR2 (e), or PHH3 (g) per unit area (WT is same as figure 3, n = 5; p53−/−, n = 4). Colored asterisks indicate the statistical significance of the difference of the same colored bars of the graph, with black asterisks indicating the total. Data are presented as mean ± SD. n.s., not significant. (h) Representative images of WT and p53−/− cortices at P1 stained for CTIP2 (red), CUX1 (green) and DAPI (blue). Scale bar: 50 μm. Note no obvious defects in neurogenesis and laminar organization in the p53−/− cortex compared to the WT control. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.

Supplementary Figure 4 No microcephaly, progenitor loss or delocalization, apoptosis, or neuronal loss in Ift88−/− brain lacking primary cilia.

(a) Representative whole mount images of E15.5 WT and conditional Ift88−/− brains. Scale bar: 0.5 mm. (b) Representative images of E15.5 WT and Ift88−/− cortices stained for ARL13B (green), a primary cilium marker, and DAPI (blue). High magnification images of the VZ surface are shown at the bottom. Note the lack of primary cilia at the VZ surface of Ift88−/− cortices compared with WT control. Scale bars: 50 μm (top) and 10 μm (bottom). (c) Representative images of E15.5 WT and Ift88−/− cortices stained for CASP3 (green) and DAPI (blue). Scale bar: 50 μm. (d, e) Representative images of E15.5 WT and Ift88−/− cortices stained for PAX6 (green, d), an RGP marker, or TBR2 (red, e), an IP marker, and DAPI (blue). Note no loss or delocalization of RGPs or IPs in Ift88−/− cortices compared with WT control. Scale bars: 50 μm. (f) Representative images of P1 WT and Ift88−/− cortices stained for CTIP2 (red), a deep layer neuron marker, CUX1 (green), a superficial layer neuron marker, and DAPI (blue). Note that there is no obvious neuronal loss in Ift88−/− cortices compared to WT control. Scale bar: 50 μm.

Supplementary Figure 5 Sas-4 deletion leads to subtle defects in neuronal distribution in the cortex.

(a) Quantification of the percentage of CUX1+ neurons in P14 WT and Sas-4−/− p53−/− cortices, which are divided into 5 bins from the pia to the white matter. Note a small but significant increase of CUX1+ neurons in the bin 4 of the Sas-4−/− p53−/− cortex. (WT, 3,150 CUX1+ cells from 5 brains; Sas-4−/− p53−/−, 3,062 CUX1+ cells from 5 brains). Data are presented as mean ± SD of individual brains. **, P < 0.01. (b) Representative images of P14 WT and Sas-4−/− p53−/− cortices stained for FOXP2 (green) and SATB2 (red), and with DAPI (blue). Note no obvious difference in the number or distribution of FOXP2+ and SATB2+ neurons in the cortex. Scale bar: 100 μm. (c) Quantification of the number of FOXP2+ and SATB2+ neurons in the cortex (WT, n = 3 brains; Sas-4−/− p53−/−, n = 3 brains). Data are presented as mean ± SD. n.s., not significant. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.

Supplementary Figure 6 Sas-4 deletion leads to a small heterotopia in the posterior dorsomedial region of the cortex.

Representative images of the posterior sections of P1 WT, Sas-4−/− p53−/− and Ift88−/− brains stained for CUX1 (green) and CTIP2 (red), and with DAPI (blue). High magnification images of the dorsolateral (area 1) and dorsomedial (area 2) regions of the cortex are shown at the bottom. Note the existence of a small heterotopia of CUX1+ neurons (broken line) in the deep layers of the Sas-4−/− p53−/− cortex. Scale bars: 500 μm (top) and 50 μm (bottom).

Supplementary Figure 7 Primary cilia loss leads to hydrocephalus.

Representative images of the anterior and posterior sections of P14 WT, Sas-4−/− p53−/− and Ift88−/− brains stained for DAPI (blue). Asterisks indicate hydrocephalus. Scale bar: 2.5 mm.

Supplementary Figure 8 Sas-4 deletion leads to p53 expression preferentially in proliferative progenitors.

(a, c) Representative images of E13.5 Sas-4−/− cortices stained with the antibodies against p53 (green) and Ki67 (red, a) or PAX6 (red, c) and DAPI (blue). High magnification images of the VZ (broken lines, 1) and CP (broken lines, 2) are shown to the right. Arrowheads indicate the double positive cells for p53 and Ki67 or PAX6. Note that virtually all p53 expressing cells are positive for Ki67 (a) and a majority of them are positive for PAX6 (c). Scale bars: 50 μm (left) and 10 μm (right). (b, d) Quantification of the percentage of p53+ cells that co-express Ki67 (b) (400 p53+ cells from 3 brains) or PAX6 (d) (391 p53+ cells from 3 brains) per unit area. Data are presented as mean ± SD of individual brains.

Supplementary Figure 9 Sas-4 deletion leads to delocalization of RGPs from the VZ.

(a) Representative images of E15.5 WT and Sas-4−/− p53−/− cortices stained for PAX6 (green) and SOX2 (red), two transcription factors expressed in RGPs, and with DAPI (blue). High magnification images of the VZ (area 1) and the IZ (area 2) are shown to the right. Note the loss of PAX6+;SOX2+ cells in the VZ and the existence of a large population of PAX6+;SOX2+ cells outside of the VZ in the Sas-4−/− p53−/− cortex. Scale bar: 50 μm (left) and 20 μm (right). (b) Quantification of the percentage of PAX6+ cells that are SOX2+. (WT, 1,388 PAX6+ cells from 3 brains; Sas-4−/− p53−/−, 1,484 PAX6+ cells from 3 brains). Data are presented as mean ± SD of individual brains. (c) Representative images of E15.5 WT and Sas-4−/− p53−/− cortices stained for PAX6 (red) and BLBP (green), a bone fide RGP marker, and with DAPI (blue). High magnification images of the VZ (area 1) and IZ (area 2) are shown to the right. Note that similar to those in the VZ, the delocalized PAX6+ cells in the SVZ and IZ of the Sas-4−/− p53−/− cortex are positive for BLBP. Scale bars: 50 μm (left) and 10 μm (right). (d) Quantification of the percentage of PAX6+ cells that are BLBP+. (WT, 1,359 PAX6+ cells from 3 brains; Sas-4−/− p53−/−, 1,511 PAX6+ cells from 3 brains). Data are presented as mean ± SD of individual brains. n.s., not significant. (e) Quantification of the percentage of PAX6+;BLBP+ cells in the extra-VZ of the Sas-4−/− p53−/− cortex that are unipolar, multipolar without a basal process (w/o BP) or multipolar with a basal process (w/ BP) in morphology based on BLBP labeling (n=253 PAX6+;BLBP+ cells). Example images of individual cells are shown to the right. Scale bar: 5 μm. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.

Supplementary Figure 10 Sas-4 deletion using Emx1-cre results in similar phenotype in microcephaly and progenitor delocalization and loss in the cortex.

(a) Representative whole mount images of E15.5 WT, Sas-4−/− and Sas-4−/− p53−/− brains generated with Emx1-Cre. Note the severe microcephaly of the Sas-4−/− but not Sas-4−/− p53−/− brain. Scale bar: 1 mm. (b) Quantification of the telencephalic area of Sas-4−/− and Sas-4−/− p53−/− mice relative to WT controls (WT, n = 4 brains; Sas-4−/−, n = 4 brains; Sas-4−/− p53−/−, n = 4 brains). Data are presented as mean ± SD. **, P < 0.01; n.s., not significant. (c) Representative images of E15.5 WT, Sas-4−/− and Sas-4−/− p53−/− cortices (broken lines) stained for PAX6 (green) or TBR2 (red) and with DAPI (blue). Double-headed arrows indicate RGPs and IPs in the VZ and the SVZ, respectively, and angled arrows indicate displaced RGPs and IPs outside the VZ and the SVZ, respectively. Note the loss of progenitors in the Sas-4−/− cortex and the rescue of delocalized progenitors in the Sas-4−/− p53−/− cortex. Scale bars: 500 μm (top), 50 μm (middle) and 50 μm (bottom). (d, e) Quantification of the number of PAX6+ (d) and TBR2+ (e) cells in the VZ and extra-VZ (WT, n = 4; Sas-4−/−, n = 3; Sas-4−/− p53−/−, n = 4). Colored asterisks indicate the statistical significance of the difference between the same colored bars of the graph and black asterisks indicate the total. Data are presented as mean ± SD. *, P < 0.05; **, P < 0.01; n.s., not significant. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.

Supplementary Figure 11 Sas-4 deletion results in a progressive loss of centrioles/centrosomes.

(a) Representative images of the VZ surface (top) or a region away from the VZ surface (Extra-VZ, bottom) in WT and Sas-4−/− cortices at E12.5, E13.5, E14.5 and E15.5 stained for PCNT (green), a centrosomal marker, ARL13B (red), a primary cilium marker, and DAPI (blue). Scale bar: 10 μm. (b) Quantification of the number of PCNT puncta (left) and primary cilia (right) relative to the number of nuclei at the VZ surface (top) and in the Extra-VZ (bottom) in WT and Sas-4−/− cortices at E12.5 (n = 4), E13.5 (n = 4), E14.5 (n = 2) and E15.5 (n = 4). *, P < 0.05; **, P < 0.01. Data are presented as mean ± SD. Note that while the loss of PCNT puncta and primary cilia in the Extra-VZ is obvious at E12.5, the loss of primary cilia at the VZ surface is not obvious until E14.5. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.

Supplementary Figure 12 Delocalized PAX6+ cells largely lack a pia-reaching basal process.

(a) Representative images of E15.5 WT and Sas-4−/− p53−/− cortices stained for PAX6 (green) and with DiI (red) applied at the pia. Arrowhead indicates an extra-VZ PAX6+ cell labeled by DiI. Note while the PAX6+ cells in the VZ of the WT or Sas-4−/− p53−/− cortex are readily labeled by DiI, the PAX6+ cells in the extra-VZ of the Sas-4−/− p53−/− cortex are rarely labeled. Scale bar: 20 μm. (b) Quantification of the percentage of PAX6+;DiI+ cells that are in a bipolar, unipolar or multipolar morphology. (WT, VZ, n = 65 cells; Sas-4−/− p53−/−, VZ, n = 70 cells; Extra-VZ, n = 40 cells). Example images are shown to the right. Scale bar: 10 μm. (c) Representative images of an E15.5 Sas-4−/− p53−/− cortex with in utero intraventricular injection of EGFP-expressing retrovirus (green) at E11.5 and stained for PAX6 (red) and P-VIMENTIN (white), and with DAPI (blue). High magnification images (broken lines) of mitotic PAX6-expressing cells outside the VZ (arrowheads) are shown to the right. Note that Extra-VZ PAX6-expressing cells in mitosis do not possess a prominent pia-directed radial glial process, indicating that they are not oRGs. Scale bars: 50 μm (left) and 10 μm (right).

Supplementary Figure 13 Sas-4 deletion leads to an increase in the number of proliferative cells but no change in cell cycle exit, consistent with a prolonged mitosis.

(a) Representative images of E15.5 WT and Sas-4−/− p53−/− cortices stained for Ki67 (green) and BrdU (red) administrated 24 hours before, and with DAPI (blue). High magnification images of the regions (broken lines) are shown at the bottom. Arrowheads indicate the cells that are BrdU+ but not Ki67+, i.e. cells that have exited the cell cycle. Scale bars: 50 μm (top) and 20 μm (bottom). (b) Quantification of the number of Ki67+ proliferative cells (WT, n = 3 brains; Sas-4−/− p53−/− n = 3 brains). Data are presented as mean ± SD. *, P < 0.05. (c) Quantification of the percentage of cells that exit the cell cycle. (WT, 1,159 BrdU+ cells from 3 brains; Sas-4−/− p53−/−, 1,492 BrdU+ cells from 3 brains). Data are presented as mean ± SD of individual brains. n.s., not significant. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.

Supplementary Figure 14 Sas-4 deletion does not lead to obvious aneuploidy, multipolar spindle formation or DNA damage in the cortex.

(a) Representative images of nuclei from superficial layer cells of P1 WT (p53−/−) and Sas-4−/− p53−/− cortices stained for three different chromosomes (Chs) using FISH: Ch 12 (green), Ch 16 (magenta) and Ch 17 (orange). Broken lines indicate the contour of individual nuclei. Scale bar: 5 μm. (b) Quantification of the percentage of cells that are disomic for all three chromosomes (WT, 224 cells from 3 brains; Sas-4−/− p53−/−, 248 cells from 3 brains). Data are presented as mean ± SD of individual brains. n.s., not significant. (c) Representative images of metaphase (top) and anaphase (bottom) cells from E15.5 WT and Sas-4−/− p53−/− cortices stained for tyrosinated α-tubulin (Tyr-TUB) (green) to show the mitotic spindle and with DAPI (blue). Arrowheads indicate spindle poles. Note mitotic cells in Sas-4−/− p53−/− cortices form bipolar spindles. Scale bar: 2 μm (d) Quantification of the percentage of mitotic spindles that are bipolar or multipolar (WT, n = 112 cells; Sas-4−/− p53−/−, n = 61 cells). (e) Western blot analysis of γ-H2AX expression from E15.5 control (Sas-4+/− p53−/−) and Sas-4−/− p53−/− cortical brain lysates. Note no increase in the expression of γ-H2AX upon SAS-4 removal (arrowhead), indicating no increase in DNA damage. (f) Representative images of E15.5 control and Sas-4−/− p53−/− cortices stained for P-VIMENTIN (red) and γH2AX (green), and with DAPI (blue). High magnification images of mitotic cells (arrowheads) are shown to the right. Scale bars: 50 μm (left) and 20 μm (right). Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.

Supplementary Figure 15 Ectopic self-renewing cells in the extra-VZ express BLBP.

(a) Representative images of E15.5 WT and Sas-4−/− p53−/− cortices administrated with sequential BrdU (red) and EdU (white) and stained for BLBP (green) and with DAPI (blue). High magnification images of the VZ of the WT cortex and the IZ of the Sas-4−/− p53−/− cortex (broken lines) are shown at the bottom. Note that similar to those in the VZ of the WT cortex, self-renewing cells that incorporate both BrdU and EdU in the IZ of the Sas-4−/− p53−/− cortex are BLBP+ (arrows), suggesting that they are ‘RGPs’ in nature. Scale bars: 50 μm (top) and 20 μm (bottom). (b,c) Quantification of the percentage (b) and distribution (c) of BrdU+;EdU+ cells that are BLBP+ (WT, 238 BrdU+;EdU+ cells and 199 BrdU+;EdU+;BLBP+ cells from 3 brains; Sas-4−/− p53−/−, 262 BrdU+;EdU+ cells and 199 BrdU+;EdU+;BLBP+ cells from 3 brains). Data are presented as mean ± SD of individual brains. **, P < 0.01. Individual P values and degrees of freedom are available in the Supplementary Methods Checklist.

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Insolera, R., Bazzi, H., Shao, W. et al. Cortical neurogenesis in the absence of centrioles. Nat Neurosci 17, 1528–1535 (2014). https://doi.org/10.1038/nn.3831

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