Lineage specificity of primary cilia in the mouse embryo

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.

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).

Developmental Biology Program, Sloan Kettering Institute, Memorial Sloan Kettering Cancer Center, 1275 York Avenue New York, New York 10065, USA,

Fiona K. Bangs, Nadine Schrode, Anna-Katerina Hadjantonakis & Kathryn V. Anderson

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.

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. (A–C) 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. (G–L) 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, (A–C) 50 μm; (D–F) 5 μm; (G–L) 50 μm, (M–O) 10 μm.

(A–D) Antibody staining for γ-tubulin (green, A) and acetylated tubulin (red, B) showed TS cells, marked by Eomes expression (magenta, C), lack primary cilia. (E–H) 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. (J–M) 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. (A–D) 5 μm; (E–H) 7 μm; (J–M) 7 μm.

(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. (G–K) 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. (L–S) 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; (G–K) 2 μm; (L–S) 5 μm.

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.

(A–F) 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). (I–K) 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 (L–N). (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). (Q–T) 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 = (A–H) 5 μm; (I–N) 5 μm; (O, P) 2 μm (Q–T) 30 μm.

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)

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)

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)

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

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)

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

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