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Lineage specificity of primary cilia in the mouse embryo

Abstract

Primary cilia are required for vertebrate cells to respond to specific intercellular signals. Here we define when and where primary cilia appear in the mouse embryo using a transgenic line that expresses ARL13B–mCherry in cilia and Centrin 2–GFP in centrosomes. Primary cilia first appear on cells of the epiblast at E6.0 and are subsequently present on all derivatives of the epiblast. In contrast, extraembryonic cells of the visceral endoderm and trophectoderm lineages have centrosomes but no cilia. Stem cell lines derived from embryonic lineages recapitulate the in vivo pattern: epiblast stem cells are ciliated, whereas trophoblast stem cells and extraembryonic endoderm (XEN) stem cells lack cilia. Basal bodies in XEN cells are mature and can form cilia when the AURKA–HDAC6 cilium disassembly pathway is inhibited. The lineage-dependent distribution of cilia is stable throughout much of gestation, defining which cells in the placenta and yolk sac are able to respond to Hedgehog ligands.

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Figure 1: Primary cilia arise in the postimplantation epiblast.
Figure 2: Primary cilia are restricted to embryonic lineages in late gastrulation stage embryos.
Figure 3: Extraembryonic lineages of the placenta and yolk sac lack cilia at E14.5.
Figure 4: Embryo-derived stem cells recapitulate the cilium status of embryonic lineages.
Figure 5: Components of the cilium disassembly pathway are highly expressed in XEN cells.
Figure 6: XEN and visceral endoderm cells form cilia when the cilium disassembly pathway is inhibited.

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Acknowledgements

We thank H. Bazzi and S. Goetz for comments on the manuscript and M. Distinti for help with manuscript preparation. We thank the MSKCC Mouse Genetics Facility for help making the transgenic mice, and the Molecular Cytology Facility and the Rockefeller University Bio-Imaging Resource Center for help with imaging. This work was supported by National Institutes of Health grants NS044385 and HD035455 to K.V.A., a NYSTEM postdoctoral fellowship to F.K.B., NYSTEM grant 029568 to A-K.H. and an MSKCC Cancer Center Support Grant (P30 CA008748).

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F.K.B. and K.V.A. designed the experiments and wrote the article. F.K.B. carried out the experiments. N.S. and A-K.H. provided advice on stem cell derivation and culture. A-K.H. provided reagents and mice.

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Correspondence to Kathryn V. Anderson.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 The ARL13B-mCherry transgene does not affect Shh signalling, rescues the phenotype of Arl13b mutants and reproduces cilia labelling with the ARL13B antibody.

Overexpression of ARL13B has been shown to promote formation of long cilia in cultured cells4. Primary cilia in mesenchymal cells of ARL13B-mCherry homozygous embryos were 37% longer (2.43 ± 0.56 μm) than in non-transgenic embryos (1.77 ± 0.34 μm, p ≤ 0.0001 from t-test, n = 50 cilia from 3 wild type and 3 transgenic embryos). Despite this, Shh-dependent neural patterning remained normal. (AC) Neural patterning in e10.5 ARL13B-mCherry homozygous embryos. Shh-dependent neural progenitor markers were expressed normally, including (A) FoxA2 (red), (B) Islet1 (green), (C) Pax7 (green). (D, E) Smo (green) was present in cilia marked with ARL13B (red) on cells of the floor plate and ventral neural tube (D) but was not present in cilia on cells of the roof plate (E). (F) Gli2 (green) was present at the tips of mesenchymal cilia marked with ARL13B. (GL) The ARL13B-mCherry transgene rescues the Arl13b mutant phenotype. Transverse sections of e10.5 ARL13bhnn/+; Tg/Tgneural tube shows the normal pattern of ventral neural cell types: floor plate cells express FoxA2 (red), V3 interneuron progenitors express Nkx2.2 (green) (G), motor neurons express Islet1 (green) and motor neuron progenitors express Olig2 (red) (J). In contrast, Arl13bhnn/hnnmutant embryos without the transgene had reduced floor plate and V3 progenitors (H) and expanded motor neurons (K), as previously described3. Neural pattern was rescued in Arl13bhnn/hnn mutants carrying ARL13B-mCherry transgene (compare I to H and L to K). (M) 72 cell blastocyst with γ-tubulin labelled centrosomes (green) not associated with acetylated α-tubulin (red) positive axonemes indicating puncta observed with ARL13B-mCherry are not cilia (Representative image selected from 9 embryos). Acetylated α-tubulin stains mid-bodies. (N and O) Antibody staining of ARL13B (red, N) or IFT88 (red, O) in Centrin2-GFP embryos (green) at e6.0 showed primary cilia (arrows) on epiblast cells but not on centrosomes on visceral endoderm (ve) cells (arrow heads; 0/659 cells from 5 embryos). Dotted line delineates epiblast (above) and visceral endoderm (below). High background staining in the visceral endoderm is an inherent technical problem with antibody staining of embryos at this stage due to high levels of endogenous IgG in visceral endoderm cells. Nuclei stained with DAPI (blue). Scale bars, (AC) 50 μm; (DF) 5 μm; (GL) 50 μm, (MO) 10 μm.

Supplementary Figure 2 Antibody staining confirms that TS cells, XEN cells and XEN-derived visceral endoderm-like cells lack primary cilia.

(AD) Antibody staining for γ-tubulin (green, A) and acetylated tubulin (red, B) showed TS cells, marked by Eomes expression (magenta, C), lack primary cilia. (EH) Antibody staining for centrosome marker γ-tubulin (green, E) and ARL13B (red, F) show XEN cells, marked by Sox17 expression (magenta, G), lack primary cilia. (I) By Western blot, wild-type XEN cells (Lane 1) and mouse embryonic fibroblasts (MEFs) (lane 2) do not express mCherry; ARL13B transgenic MEFs (lane 3) express an mCherry band (arrow) at 88 kDa, the size of ARL13B (60 kDa) plus mCherry (28 kDa). This band is also detected in transgenic XEN cells (lane 4 and 5). Higher molecular weight band is non-specific. (JM) XEN cells differentiated into visceral endoderm lack primary cilia. Addition of CHIR99201 upregulates FoxA2 (Magenta, J) and E-Cadherin (magenta, L), demonstrating XEN cells have differentiated into visceral endoderm, however no cilia, marked by expression of acetylated α-tubulin (red), form; centrosomes marked with γ-tubulin (green) (K, M). DAPI stains nuclei in blue. (AD) 5 μm; (EH) 7 μm; (JM) 7 μm.

Supplementary Figure 3 Lack of cilia on XEN cells is not due to lack of RFX3 expression or negative regulation via CP110.

(A, B) RFX3 (green) is expressed at higher levels in epiblast cells than visceral endoderm cells at e6.5. (C, D) RFX3 is expressed in mESC and (E, F) XEN cell nuclei. (GK) Localization of Cep164, Ninein, TTBK2, IFT88, CP110 (green) in ciliated EpiSCs stained with γ-tubulin (red) to mark the centrosome and acetylated α-tubulin (magenta) to mark the axoneme. (LS) Knockdown of CP110 (green, L) leads to formation of long centrioles in 16.6 ± 7.7% of cells (65/503 cells from 3 independent experiments), marked with γ-tubulin (red) and acetylated α-tubulin (magenta) and Centrin2-GFP (green, N) in XEN cells. These long structures do not express the cilia markers IFT88 (green, P) or ARL13B (green, R). Centrioles and the localization of IFT88 are unaffected in scramble treated controls (M, O, Q, S). DAPI stains nuclei in blue. Scale bars, (A, B) 20 μm; (C, D) 5 μm; (E, F) 7 μm; (GK) 2 μm; (LS) 5 μm.

Supplementary Figure 4 Plk1, APC/C ceramide, VHL and HIFα, which have been reported to regulate ciliogenesis in specific cell types, do not regulate cilia formation in XEN cells.

Polo like kinase 1 (Plk1) is recruited to the centrosome via PCM1 and activated by AurkA, where it activates HDAC6, promoting cilia disassembly29. 24 h treatment of XEN cells with 1 μM Plk1 inhibitor BI2536 did not cause cilia formation in XEN cells (0/308 cells, DMSO controls 0/301 cells, 1 independent experiment), despite formation of monopolar spindles, which are associated with loss of Plk1 activity. N-nervonoyl d-erythro sphingosine (C24:1) ceramide is required for ciliogenesis in MDCK cells and in human ES cell derived neural progenitors to sequester aPKC to the apicolateral cell membrane preventing it from activating AURKA, leading to cilium disassembly30. Exposure to 2 μM or 20 μM of C24:1 ceramide for 24 h did not induce ciliogenesis in XEN cells (0/281 cells, DMSO controls 0/322 cells, 1 independent experiment). APC/C and Cdc20 localize to the basal body in human epithelial cells and are required for cilia disassembly before anaphase. This is mediated by APC/C dependent degradation of Nek1, which is essential for maintenance of axonemal microtubule stability and integrity31. Treatment of XEN cells for 24 h with 20 μM APC inhibitor proTAME did not induce cilia formation in these cells (0/326 cells, DMSO controls 0/345 cells, 1 independent experiment). VHL is required for cilia stability in renal cysts specifically when GSK3β is inactive35 (A) Western blot shows that VHL protein is present at equivalent levels in XEN cells and EpiSCs (2 independent experiments). Thus VHL protein is not limiting for cilia formation in XEN cells, suggesting that the pVHL-GSK3β ciliary-maintenance signalling network may only be active in renal tissues. (B, C) 24 h treatment of XEN cells with 1 μM FM19G11, which blocks the function of both HIF1α and HIF2α (IC50 = 80 nM37) but does not cause cilia formation (0/284 cells; DMSO 0/278 cells; from 2 independent experiments), marked with IFT88 (green), acetylated α-tubulin (magenta) and centrosomes γ-tubulin (red). NEDD9 and AURKA are therefore activated in XEN cells by a HIF-independent pathway. Nuclei stained with DAPI (blue). Scale bar = (B, C) 5 μm.

Supplementary Figure 5 AurkA and HDAC6 inhibitors and siRNA controls.

(AF) Phosphorylated AurkA (green) is detected at the spindle pole marked with γ-tubulin (red) in mitotic cells, arrowed (A, C) but is lost upon treatment with Alisertib (B, D). (E, F) Total AurkA (green) is still detected at the centrosome, marked with pericentrin (PCNT, red) in Alisertib treated cells (F). (G, H) Following Alisertib treatment, ciliated XEN cells continue to express XEN cell markers including Sox17 (red). (IK) siRNA knock down of AurkA reduces levels of AurkA (green) at the centrosome, marked with PCNT (red), compared to control cells treated with scrambled siRNA (LN). (O, P) Treatment of XEN cells with 5 μM HDAC6 inhibitor Tubacin for 72 h causes formation of primary cilia, marked with IFT88 (O, green), ARL13B (P, green), and acetylated α-tubulin (magenta), that project from a centrosome marked with γ-tubulin (red; 22/573 cells from 3 independent experiments). (QT) Culture of e7.5 embryos for 12 h in 5 μM Tubacin causes stabilization of microtubules, marked by upregulation of acetylated α-tubulin (red). Average pixel intensity of acetylated α-tubulin was 4.6 times higher in Tubacin-treated embryos than in DMSO controls. Nuclei stained with DAPI (blue). Scale bar = (AH) 5 μm; (IN) 5 μm; (O, P) 2 μm (QT) 30 μm.

Supplementary information

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3D reconstruction of confocal imaging of a 32 cell blastocyst expressing ARL13B-mCherry.

Centrioles marked with Centrin2-GFP (green) are present in ICM and TE cells but primary cilia are not present in the embryo at this stage. (MOV 3618 kb)

3D reconstruction of confocal imaging of a 72 cell blastocyst expressing ARL13B-mCherry and Centrin2-GFP.

On rare occasions (2% of cells), ARL13B-mCherry expression can be detected adjacent to centrosomes in cells of the ICM but not in trophectoderm cells. (MOV 5426 kb)

3D reconstruction of confocal imaging of an e5.5 embryo after cavitation, expressing ARL13B-mCherry and Centrin2-GFP.

Some ARL13b-mCherry expression can be detected adjacent to centrosomes in the epiblast but not the extraembryonic visceral endoderm or trophoectoderm lineages. (MOV 8249 kb)

3D reconstruction of confocal imaging of an e6.0 embryo expressing ARL13B-mCherry (red) and Centrin2-GFP.

Primary cilia are only present on epiblast cells and not on cells of the visceral endoderm. (MOV 4370 kb)

3D reconstruction of confocal imaging of the distal region and node of an 8.0 embryo.

Primary cilia expressing ARL13B-mCherry (red) are broadly distributed in both the long cilia of the node (the pit at the centre of the Video) and in cells of all three germ layers. Centrioles are marked with Centrin2-GFP (green). (MOV 19759 kb)

3D reconstruction of confocal imaging of an early bud embryo (e7.5), AFP-GFP (green) is expressed in visceral endoderm cells.

ARL13B-mCherry (red) labels primary cilia, which are only present on non-GFP expressing definitive endoderm cells. (MOV 10495 kb)

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Bangs, F., Schrode, N., Hadjantonakis, AK. et al. Lineage specificity of primary cilia in the mouse embryo. Nat Cell Biol 17, 113–122 (2015). https://doi.org/10.1038/ncb3091

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