# Actin-based deformations of the nucleus control multiciliated ependymal cell differentiation

Marianne Basso, Alexia Mahuzier, Syed Kaabir Ali, Anaïs Marty, Marion Faucourt, Ana-Maria Lennon-Duménil, Ayush Srivastava, Michella Khoury, Damaa, Alexia Bankolé, Alice Meunier, et al.

► To cite this version:

Marianne Basso, Alexia Mahuzier, Syed Kaabir Ali, Anaïs Marty, Marion Faucourt, et al. Actin-based deformations of the nucleus control multiciliated ependymal cell differentiation. Developmental Cell, 2025, 60 (5), pp.749-761. 10.1016/j.devcel.2024.11.008. hal-04739832

HAL Id: hal-04739832

https://hal.sorbonne-universite.fr/hal-04739832v1

Submitted on 11 Apr 2025

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<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
# Developmental Cell

## Actin-based deformations of the nucleus control mouse multiciliated ependymal cell differentiation

### Graphical abstract

<figure><img src="image_2.png" /></figure>

### Authors

Marianne Basso, Alexia Mahuzier, Syed Kaabir Ali, ..., Julie Plastino, Nathalie Spassky, Nathalie Delgehyr

### Correspondence

delgehyr@bio.ens.psl.eu

### In brief

Brain multiciliated cells contribute to cerebrospinal fluid flow, a process impaired in many neurological disorders. Here, Basso et al. show that at differentiation onset, actin-dependent forces deform the nucleus of the progenitor cells, promoting multiciliated cell differentiation through RB1 phosphorylation and activation of the transcriptional regulator MCIDAS.

### Highlights

* Actin, through the LINC complex, deforms the nucleus during ependymal differentiation

* Nuclear deformation triggers ependymal differentiation

* RB1 phosphorylation occurs downstream of nuclear deformation

* RB1 phosphorylation induces MCIDAS activation and ependymal differentiation

<figure><img src="image_3.png" /></figure>
Article

# Actin-based deformations of the nucleus control mouse multiciliated ependymal cell differentiation

Marianne Basso,<fnref n="1" />,<fnref n="6" /> Alexia Mahuzier,<fnref n="1" />,<fnref n="6" /> Syed Kaabir Ali,<fnref n="1" />,<fnref n="2" />,<fnref n="3" />,<fnref n="6" /> Anaïs Marty,<fnref n="3" /> Marion Faucourt,<fnref n="1" />

Ana-Maria Lennon-Duménil,<fnref n="4" /> Ayush Srivastava,<fnref n="1" /> Michella Khoury Damaa,<fnref n="1" /> Alexia Bankolé,<fnref n="5" /> Alice Meunier,<fnref n="1" />

Ayako Yamada,<fnref n="3" /> Julie Plastino,<fnref n="2" /> Nathalie Spassky,<fnref n="1" />,<fnref n="7" /> and Nathalie Delgehyr<fnref n="1" />,<fnref n="7" />,<fnref n="8" />,*

## SUMMARY

Ependymal cells (ECs) are multiciliated cells in the brain that contribute to cerebrospinal fluid flow. ECs are specified during embryonic stages but differentiate later in development. Their differentiation depends on genes such as GEMC1 and MCIDAS in conjunction with E2F4/5 as well as on cell-cycle-related factors. In the mouse brain, we observe that nuclear deformation accompanies EC differentiation. Tampering with these deformations either by decreasing F-actin levels or by severing the link between the nucleus and the actin cytoskeleton blocks differentiation. Conversely, increasing F-actin by knocking out the Arp2/3 complex inhibitor Arpin or artificially deforming the nucleus activates differentiation. These data are consistent with actin polymerization triggering nuclear deformation and jump starting the signaling that produces ECs. A player in this process is the retinoblastoma 1 (RB1) protein, whose phosphorylation prompts MCIDAS activation. Overall, this study identifies a role for actin-based mechanical inputs to the nucleus as controlling factors in cell differentiation.

## INTRODUCTION

Multiciliated ependymal cells (ECs) are located along brain ventricles. They form a protective barrier to the brain parenchyma and contribute to cerebrospinal fluid flow through the beating of their motile cilia.<fnref n="1" /> Impairment of this flow correlates with age-related dementia,<fnref n="2" /> schizophrenia,<fnref n="3" /> and hydrocephalus,<fnref n="4" /> diseases whose onset is not always understood.

In the mammalian brain, lineage tracing experiments show that ECs are sister cells of the adult neural stem cell,<fnref n="5" />,<fnref n="6" /> derived from embryonic progenitors<fnref n="7" /> but acquiring their own identity at later stages. Differentiation of multiciliated cells depends on Notch inhibition, which is required for the activation of the transcriptional regulators GEMC1 (Geminin coiled-coil domain-containing protein 1), followed by MCIDAS (multiciliate differentiation and DNA synthesis associated cell cycle protein) and other effectors.<fnref n="8" /> Both GEMC1 and MCIDAS interact with E2F (Eukaryotic 2 transcription factor) 4 or 5 and DP1 (dimerization partner1) to promote transcriptional control of multiciliated cell differentiation.⁹ Cell-cycle-related factors, such as the Cyclin Dependent Kinase (CDK) 1 and 2, are also involved in the differ-

entiation of these post-mitotic cells.¹⁰,¹¹ The cues that trigger the activation of the different factors required for differentiation are not clear.

Neural stem cell progenitors are sensitive to forces during their proliferative and differentiating stages in neurons or oligodendrocytes,¹² and it is known in stem cells in general that forces can be directly transmitted to the nucleus through the cytoskeleton.¹³ Key players in this transmission are the LINC (linkers of nucleoskeleton and cytoskeleton) complexes, which span the nuclear envelope and link the nucleoskeleton to the cytoskeleton.¹⁴ In these complexes, the inner nuclear membrane proteins, the SUN (for Sad1 and UNC-84) family, contact the lamina (nucleoskeleton) and interact with a specific domain (KASH (for Klarsicht, ANC-1, Syne homology) domain) of the outer nuclear membrane proteins (the NESPRIN (Nuclear envelope spectrin-repeat protein) family encoded by the Syne genes). NESPRINS interact with either microtubules, intermediate filaments, or actin. Actin has been shown to contribute to nuclear morphology and positioning in different systems (e.g., migrating fibroblasts¹⁵ and oocytes¹⁶). The LINC complexes and the mechanical forces transmitted by these complexes to the nucleus are involved in

<footnote n="1">¹Institut de biologie de l'Ecole normale supérieure (IBENS), Ecole normale supérieure, CNRS, INSERM, Université PSL, 75005 Paris, France</footnote>

<footnote n="2">²Laboratoire de physique de l'Ecole Normale Supérieure, Université PSL, CNRS, Sorbonne Université, Université Paris Cité, 75005 Paris, France</footnote>

<footnote n="3">³PASTEUR, Département de Chimie, Ecole Normale Supérieure, Université PSL, Sorbonne Université, CNRS, 75005 Paris, France</footnote>

<footnote n="4">⁴Institut Curie, INSERM U932, Université PSL, 75005 Paris, France</footnote>

<footnote n="5">⁵Institut Necker Enfants Malades (INEM), Université Paris Cité, CNRS, INSERM, 75015 Paris, France</footnote>

<footnote n="6">⁶These authors contributed equally</footnote>

<footnote n="7">⁷These authors contributed equally</footnote>

<footnote n="8">⁸Lead contact</footnote>

<footnote>*Correspondence: delgehyr@bio.ens.psl.eu
https://doi.org/10.1016/j.devcel.2024.11.008</footnote>
<figure><img src="image_4.png" /></figure>

many aspects of cellular functions. For example, forces partici-
pate in the orientation and shape of the nucleus,¹⁷,¹⁸ the posi-
tioning of the centrosome,¹⁹ DNA damage and repair,²⁰⁻²²
changes in gene expression,²³⁻²⁶ and the opening of nuclear
pores or channels,²⁷,²⁸ with consequences for cell cycle²⁹⁻³³ or
fate choice.³⁴⁻³⁷

In this context, we sought to identify the cues that drive
EC differentiation. We observed a connection between
actin accumulation, nuclear deformation, and differentiation.
Other results imply that retinoblastoma 1 (RB1) protein is
involved in transducing nuclear deformations to activate EC
differentiation.

RESULTS

Nuclei deform during EC differentiation

To visualize nuclear shape during EC differentiation, we stained
the lateral ventricles of mice expressing centrin2-GFP³⁸ during
the process of differentiation (post-natal day 5, P5) with lamin-
B1 antibodies. We observed a large diversity of shapes and
localizations of nuclei (Figure 1A). The centrin2-GFP allowed
us to determine the stage of EC differentiation. Cells at the sur-

<caption>**Figure 1. Nuclei deform during ependymal differentiation**</caption>

(A and G) All stainings were performed on whole-
mount lateral wall during EC differentiation (post-
natal day 5, P5) on centrin2-GFP mice. (A) Staining
of lamin-B1 (nuclear lamina, gray) and centrin2-
GFP (Cen2GFP, centrioles, green). Top view is a
maximum projection of z stacks. Side view is an
Imaris 3D projection of the above cell (orange
dashed circle) with segmented nuclei.

(B–D) Quantification of the nuclear shape on Imaris segmented nuclei of cells transfected at P1 with membrane-cherry (mb-cherry) to associate the apical surface with its nucleus. (B) Volume. (C) Pole-to-pole elongation index (prolate ellipticity). (D) Distance between the center of the nucleus and the highest centriole. Error bars represent the SEM of n = 54 progenitors (Prog), 66 differentiating (Diff), and 54 multi-basal bodies (Multi) cells from nine mice; ***p ≤ 0.001; **p ≤ 0.01 (One-way ANOVA followed by Dunn's multiple comparison). (E) Staining of H3K27me3 (gray, top) or H3K9me2 (gray, bottom), centrin2-GFP (Cen2GFP, green), and Sas6 (pro-centriole, red).

(F and G) Quantification of the fluorescence in-
tensity of H3K27me3 (F) or H3K9me2 (G) staining
in cells transfected with mb-cherry, normalized to
the mean intensity in surrounding untransfected
progenitor cells. Error bars represent the SEM of
n = 76/54 progenitors, 40/20 differentiating, and
56/30 multi-basal body cells from three mice for
H3K27me3 and H3K9me2, respectively; ***p ≤
0.001; ns, p > 0.05 (one-way ANOVA followed by
Dunn's multiple comparison). Scale bar, 5 µm.

face of the ventricle with two centrin
dots are progenitors. Subsequently, a
cloud or ring-like structure of centrin
appear near the two centrin dots, indi-

cating the formation of pro-centrioles around deuterosomes (differentiating cells). Finally, several dots (on average 50³⁹) of centrin are released from the deuterosomes and migrate toward the plasma membrane (multi-basal body cells) where they dock to form the basal bodies at the base of motile cilia.⁴⁰ In the tissue, nuclei are quite close to each other, sometimes in depth, making it challenging to associate each nucleus with its corresponding centrin staining. To solve this problem, we electroporated centrin2-GFP-expressing brains at the onset of differentiation (P1) with membrane-cherry (mb-cherry). This reporter stained the membranes of the electroporated cells, allowing the identification of which nucleus goes with which apical surface, where centrioles are located, to assess differentiation stages. In electroporated lateral walls stained for lamin B1, we noted that during the process of differentiation (P5), nuclei increased in volume (1.7× increase on average between progenitor and multi-basal body stages, Figures 1A and 1B), changed shape (becoming 2× flatter at the poles, Figures 1A and 1C) and migrated toward the plasma membrane (moving around 2 µm up on average, Figures 1A and 1D). Overall, nuclei were drastically changing during the process of ependymal differentiation.
In the literature, there are reports that mechanical forces applied to the nucleus drive chromatin re-organization and cell fate acquisition.⁴¹,⁴² We thus wondered whether the observed changes in nuclear shape and localization were accompanied by a change in chromatin organization. To assess this, we stained centrin2-GFP mouse lateral wall (electroporated at differentiation onset [P1] with mb-cherry) in the process of ependymal differentiation (P5) with Sas6 (a pro-centriole marker present during the differentiating stage only) and the heterochromatin markers H3K27me3 or H3K9me2. Similar to the response observed in cells subjected to an artificial mechanical stress,⁴³ we observed that H3K27me3 increased, whereas H3K9me2 decreased, with nuclear re-shaping and differentiation (Figures 1E-1G). Thus, the nuclei of differentiating cells are actively deforming and rearranging their chromatin.

**Actin, but not microtubules, is involved in nuclear deformation and EC differentiation**

Nuclear deformation and localization in many systems depend on the cell cytoskeleton.⁴⁴,⁴⁵ F-actin and microtubule staining on the lateral wall during EC differentiation showed that both networks undergo massive rearrangement, accumulating near the centrioles during the first steps of differentiation (Figures S1A-S1D). To explore the contribution of both F-actin and microtubules to nuclear shape change, we treated explants of lateral walls containing cells in the process of differentiation (P5) with drugs leading to the depolymerization of microtubules (nocodazole) or actin (cytochalasin-D) for 20 h. We then fixed and stained the explants for lamin B1. Cytochalasin-D perturbed the morphology of the nuclei of differentiated cells, whereas nocodazole had no obvious effect (Figure S1E). In consistent with this, interfering with the microtubule molecular motor dynein had no effect on either nuclear deformation (Figures S2A-S2C) or EC differentiation (Figures S2D-S2G). The slight effect observed in nuclear position in relation to the centrioles at the progenitor stage might be a consequence of impairment of the interkinetic movement observed in the few remaining dividing progenitor cells at P5 (most of the division happening during embryonic development⁷,⁴⁶).

Actin cables are enriched at the apical surface of differentiating cells, with long cables reaching the nuclei, suggesting that the actin network might cap the nucleus or pull on it. Interestingly, inhibiting myosin activity using blebbistatin treatment for 20 h in lateral ventricle explants bearing differentiating ECs also affected the deformation of multiciliated cell nuclei (Figure S1E). A host of different actin binding proteins are known to contribute to the actin network in differentiated multiciliated cells.⁴⁷ To assess whether F-actin accumulation during differentiation depends on the Arp2/3 complex (branched F-actin) or on formins (linear F-actin), we treated explants of lateral walls bearing differentiating ECs (P5) with inhibitors of the Arp2/3 complex (CK666) or of formins (SMIFH2) for 20 h and then fixed and stained them with phalloidin (Figures S1F and S1G). Only the treatment with CK666 decreased the accumulation of F-actin in differentiating cells, suggesting a contribution of the Arp2/3 complex to F-actin formation in ECs.

To perturb Arp2/3 complex activity in vivo, we electroporated at differentiation onset (P1) an shRNA (short hairpin RNA) against Arp3 for which the sequence was validated.⁴⁸ ARP3 is a subunit

of the Arp2/3 complex. We performed phalloidin staining at P5 on electroporated lateral walls and showed that shArp3 decreased F-actin accumulation in differentiating cells compared with control cells and relative to untransfected surrounding progenitor cells (Figures S3A and S3B). Lamin-B1 staining in cells depleted for Arp3 revealed that the nuclei did not flatten as much as in control electroporated cells and migrated less toward the apical side (Figures 2A-2C), whereas nuclear volumes remained similar to control (Figure S3C). To assess the effect of Arp3 depletion on EC differentiation, we electroporated the lateral ventricles of centrin2-GFP expressing mice with shArp3 and mb-cherry at P1 and quantified the extent of differentiation at P5 and P15. Already at P5, we observed that fewer progenitors had differentiated compared with controls (Figures 2D and 2E). We should point out that some of the differentiated cells observed at P5 might have already been engaged in the process of differentiation at the time of electroporation (P1), as differentiation is asynchronous. This is consistent with the fact that at P15, we observed a much stronger defect in EC differentiation in cells electroporated with the shArp3 compared with the control (Figures 2F and 2G). Altogether, these data suggest that branched F-actin is involved in the deformation/localization of the nuclei to promote EC differentiation.

If this is the case, we would expect that performing the inverse experiment, i.e., increasing actin polymerization, would increase nuclear deformation and EC differentiation. To test this hypothesis, we examined EC differentiation in an *Arpin* conditional knockout (cKO) mouse⁴⁹; the *Arpin* gene encodes for a negative regulator of the Arp2/3 complex so the KO has enhanced branched actin polymerization.⁵⁰ We electroporated the lateral ventricle of *Arpin* cKO mice at P1 with the Cre recombinase and the mb-cherry marker. We first confirmed that removing *Arpin* increased F-actin accumulation in differentiating ECs compared with control electroporation and relative to untransfected surrounding progenitors (Figures S3A and S3B). We also stained the electroporated lateral walls for lamin B1 and observed that increasing F-actin polymerization triggered nuclei deformation and migration toward the apical surface at earlier stages than in control cells (Figures 3A-3C). Finally, we assessed whether increasing F-actin perturbed EC differentiation by staining for centriole and deuterosome (the platform around which pro-centrioles are assembled) markers. We observed that removing *Arpin* at differentiation onset led to an increase in differentiated cells (Figures 3D and 3E) at P5. Similarly, at P15, we observed significantly more multiciliated ECs in the animals electroporated with Cre compared with the control (Figures 3F and 3G). These results suggest that an increase in Arp2/3 activity led to premature differentiation of progenitors into ECs.

**Severing the link between the nucleus and the cytoskeleton perturbs nuclear deformation and ependymal differentiation**

F-actin has many functions in cells; therefore, decreasing or increasing actin amounts could have off-target effects. To address this issue, we left the actin intact but perturbed its link to the nucleus by expressing in the cells, at the onset of differentiation (P1), a dominant negative construct composed of the KASH domain from NESPRIN1.⁵¹ This domain displaces the endogenous NESPRINS by interacting with SUN proteins but
<figure><img src="image_5.png" /><figcaption>**Figure 2. Decreasing ARP3 impairs nuclear deformation and ependymal differentiation**</figcaption></figure>

(A–G) All staining were performed on whole mount lateral walls. Wild-type (A–C) or centrin2-GFP (D and G) mice transfected at P1 with mb-cherry as a reporter and either a control plasmid (Ctrl) or an shRNA against Arp3 (shArp3).

(A) Staining of lamin B1 (gray) and FOP (centrioles, green) during EC differentiation (P5). Transfected cells, either control (top row) or shArp3 (bottom row), are circled with orange dashed lines (Transf). Top view is a maximum projection of z stacks. Side view is an Imaris 3D projection with segmented nuclei. The white dashed lines represent the plasma membrane.

(B and C) Quantification of nuclear shapes during EC differentiation on Imaris segmented nuclei as in (A). (B) Pole-to-pole elongation index. (C) Distance between the center of the nucleus and the highest centriole. Error bars represent the SEM of n = 26/ 73 progenitors, 40/29 differentiating, and 31/39 multi-basal bodies (Multi BB) transfected cells (Ctrl/shArp3) from four mice; **p ≤ 0.001; **p ≤ 0.01; ns, p > 0.05 (Mann-Whitney).

(D) Staining of centrin2-GFP (Cen2GFP, green), Sas6 (gray), mb-cherry (transfection reporter, red), and beta-catenin (βcat, blue) during EC differentiation (P5).

(E) Quantification of the percentage of transfected cells as in (D) at each stage of differentiation (Prog, progenitors; Diff, Differentiating; Multi, multi-basal bodies). Error bars represent the SEM of five experiments n = 174 (Ctrl) and 183 (shArp3) transfected cells; **p ≤ 0.01 (two-way ANOVA followed by Sidak's multiple comparisons).

(F) Staining of centrin2-GFP (Cen2GFP, green), mb-cherry (transfection reporter, red), and beta-catenin (βcat, blue) in differentiated cells (P15).

(G) Quantification of the percentage of transfected cells as in (F) remaining as progenitors (Prog), still differentiating (Diff), or differentiated in ECs (Multi). Error bars represent the SEM of four or three experiments n = 83 (Ctrl) and 93 (shArp3) transfected cells, respectively; **p ≤ 0.001 (two-way ANOVA followed by Sidak's multiple comparisons). Scale bar, 5 µm.

cannot interact with the cytoskeleton. We first assessed whether perturbation of the LINC complex changed F-actin organization during ependymal differentiation. We stained lateral ventricles electroporated at P1 with the KASH domain or a control plasmid and mb-cherry with phalloidin at P5. We did not observe changes in the F-actin organization compared with the control electroporated cells (Figures S3A and S3B) and relative to un-transfected surrounding progenitor cells. However, considering the complexity of actin staining in differentiating cells, we cannot rule out some local actin rearrangement, especially because there are reports in other systems of crosstalk between the LINC complex and the actin cytoskeleton.⁵²,⁵³ We also stained for lamin B1 and observed that the nuclei of differentiating cells (P5) expressing the KASH flattened less than control nuclei and remained in the depth of the tissue (Figures 4A–4C). Thus, disrupting the LINC complex was sufficient to perturb nuclear morphology without drastically affecting F-actin polymerization. We then assessed how the cells differentiate in the absence of

functional LINC complex. At P5, when the cells are differentiating, expressing the KASH domain already slightly impaired differentiation (Figures 4D and 4E), and this effect was even more pronounced at P15 (Figures 4F and 4G). Altogether, these experiments show that severing the link between the nucleus and the cytoskeleton is sufficient to perturb nuclear morphology and EC differentiation without affecting drastically actin organization.

To extend this observation and identify which LINC complex components were involved, we stained the differentiating tissue with antibodies directed against LINC complex proteins. Among them, we saw that NESPRIN1 and 2 were expressed during differentiation (Figure 5A). We observed that NESPRIN1 accumulated at an early stage of differentiation at the centrosome/growing centrioles and was enriched at the nuclear envelope at later stages of differentiation, whereas NESPRIN2 was recruited to the nuclear envelope very early in the differentiation process. We quantified this recruitment in an *in vitro* culture of differentiating progenitor cells⁵⁴⁻⁵⁶ as the background was less pronounced than *in vivo*
<figure><img src="image_6.png" /></figure>

(Arpin<sup>KO/lox</sup>; Cre) experiments, *n* = 314 (Ctrl) and 151 (Arpin<sup>KO/lox</sup>; Cre) transfected cells; \* *p* ≤ 0.05 (two-way ANOVA followed by Sidak's multiple comparisons). Scale bar, 5 µm.

(Figures S4A–S4D). This analysis confirmed the appearance of NESPRIN2 at the nuclear envelope earlier than NESPRIN1 and the accumulation of NESPRIN1 at the centrosome during the process of differentiation. Intriguingly, we observed an increase in actin levels after NESPRIN1 depletion, using a validated shRNA sequence against Syne1<sup>57</sup> (Figures S4E–S4G), in both differentiating (Figures S3A and S3B) and progenitor cells (Figures S3D and S3E). Because it happened before NESPRIN1 was recruited to the nuclear envelope, it might be dependent on the non-nuclear NESPRIN1. In accordance with the actin levels, NESPRIN1 depletion led to a premature flattening and relocalization of the nucleus (Figures S5A–S5C) and accelerated ependymal differentiation (Figures S5D–S5G). On the other hand, depleting NESPRIN2 using a validated shRNA sequence against Syne2<sup>57</sup> (Figures S4H and S4I) had a similar effect as expressing the KASH domain: no drastic effect on actin organization (Figures S3A and S3B), and both nuclear deformation/relocalization (Figures 5B–5D) and ependymal differentiation (Figures 5E and 5F) impairment. Altogether, these

experiments show that severing the NESPRIN2/cytoskeleton interaction is sufficient to perturb the nuclear morphology and EC differentiation without drastically affecting actin organization.

## Confining the cell and its nucleus induces EC differentiation

To more directly assess whether nuclear deformation triggered ependymal differentiation, we applied an artificial mechanical load on the nucleus. Because it is challenging to perform mechanical loading on cells *in vivo*, we used our cell culture model of ependymal differentiation. We first confirmed that the nuclei deformed during *in vitro* differentiation. To do so, we stained centrin2-GFP cells 5 days (D5) after serum withdrawal (differentiation onset, D0) for lamin B1 (lamina) and Sas6 (pro-centrioles). We then analyzed the nuclear shape at different stages of differentiation as determined by the pattern of centrin2-GFP/Sas6. We observed that *in vitro*, the nuclei increased in size (1.3×) and flattened (1.4×) during ependymal

<caption>**Figure 3.** Increasing Arp2/3 complex activity facilitates nuclear deformation and ependymal differentiation</caption>

(A–G) All staining were performed on whole mount lateral walls. Arpin<sup>lox/lox</sup> transfected at P1 with mb-cherry as a reporter and a control plasmid (Ctrl) or Arpin<sup>KO/lox</sup> mice transfected at P1 with mb-cherry as a reporter and a plasmid coding for the Cre recombinase (Arpin<sup>KO/lox</sup>; Cre).

(A) Staining of lamin B1 (gray) and FOP (green) during EC differentiation (P5). Transfected cells, either Ctrl (top row) or Arpin<sup>KO/lox</sup>; Cre (bottom row) are circled with orange dashed lines (Transf.). Top view is a maximum projection of z stacks. Side view is an Imaris 3D projection with segmented nuclei. The white dashed lines represent the plasma membrane.

(B and C) Quantification of nuclear shapes during the different steps of EC differentiation on Imaris segmented nuclei of cells as in (A). (B) Pole-to-pole elongation index. (C) Distance between the center of the nucleus and the highest centriole. Error bars represent the SEM of *n* = 48/53 progenitors, 50/31 differentiating, and 40/40 multi-basal bodies (Multi BB) cells (Ctrl/Arpin<sup>KO/lox</sup>; Cre) from seven mice in Ctrl and eight mice in Arpin<sup>KO/lox</sup>; Cre; \*\* *p* ≤ 0.01; \* *p* ≤ 0.05; ns, *p* > 0.05 (Mann-Whitney).

(D) Staining of FOP (green), Deup1 (deuterosomes, gray) and mb-cherry (transfection reporter, red) during EC differentiation (P5).

(E) Quantification of the percentage of transfected cells as in (D) at each stages of differentiation. Error bars represent the SEM of five (Ctrl) or four (Arpin<sup>KO/lox</sup>; Cre) experiments, *n* = 178 (Ctrl) and 145 (Arpin<sup>KO/lox</sup>; Cre) transfected cells; Prog, progenitors; Diff, differentiating; and Multi, multi-basal body cells. \*\*\* *p* ≤ 0.001; ns, *p* > 0.05 (two-way ANOVA followed by Sidak's multiple comparisons).

(F) Staining of FOP (green) and mb-cherry (transfection reporter, red) in differentiated cells (P15).

(G) Quantification of the percentage of transfected cells as in (F) remaining as progenitors (Prog), still differentiating (Diff) or differentiated in EC (Multi). Error bars represent the SEM of six (Ctrl) and five
<figure><img src="image_7.png" /></figure>

Lamin B1 FOP Transf

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /></figure>

<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>**Figure 4.** Severing the link between the nucleus and the cytoskeleton impairs nuclear deformation and ependymal differentiation</figcaption></figure>

(A–G) All staining were performed on whole mount lateral walls. Wild-type (A–C) or centrin2-GFP (D–G) mice transfected at P1 with mb-cherry as a reporter and either a control plasmid (Ctrl) or the KASH domain (dominant negative of the LINC complex, KASH).

(A) Staining of lamin B1 (gray) and FOP (green) during EC differentiation (P5). Transfected cells, either Ctrl (top row) or KASH (bottom row), are circled with orange dashed lines (Transf). Top view is a maximum projection of z stacks. Side view is an Imaris 3D projection with segmented nuclei. The white dashed lines represent the plasma membrane.

(B and C) Quantification of the nuclear shape during the different steps of EC differentiation on Imaris segmented nuclei as in (A). (B) Pole-to-pole elongation index. (C) Distance between the center of the nucleus and the highest centriole. Error bars represent the SEM of *n* = 29/21 progenitors, 30/26 differentiating, and 23/22 multi-basal bodies (Multi BB) transfected cells (Ctrl/KASH) from three mice; \*p < 0.05; ns, *p* > 0.05; \*\*p ≤ 0.01 (Mann-Whitney).

(D) Staining of centrin2-GFP (Cen2GFP, green), Sas6 (gray), mb-cherry (transfection reporter, red) and beta-catenin (βcat, blue) during EC differentiation (P5).

(E) Quantification of the percentage of transfected cells as in (D) at each stages of differentiation. Error bars represent the SEM of five experiments; *n* = 131 (Ctrl) and 189 (KASH) transfected cells; Prog, progenitor; Diff, differentiating; Multi, multi-basal body cells. \*\*p ≤ 0.01; \*p ≤ 0.05; ns, *p* > 0.05 (two-way ANOVA followed by Sidak's multiple comparisons).

(F) Staining of centrin2-GFP (Cen2GFP, green), mb-cherry (transfection reporter, red), and beta-catenin (βcat, blue) in differentiated cells (P15).

(G) Quantification of the percentage of transfected cells as in (F) remaining as progenitors

(Prog), still differentiating (Diff), or differentiated (Multi). Error bars represent the SEM of six (Ctrl) or three (KASH) experiments, *n* = 148 (Ctrl) and 86 (KASH) transfected cells; \*\*\*p ≤ 0.001 (two-way ANOVA followed by Sidak's multiple comparisons). Scale bar, 5 µm.

differentiation (Figures 6A–6C) compared with *in vivo*. Additionally, we observed that H3K27me3 increased during *in vitro* differentiation, whereas H3K9me2 decreased (Figures 6D–6F). We also performed shRNA against *Arp3* and *Syne2* *in vitro* and observed that their depletion led to an impairment of EC differentiation (Figures 6G and 6H). Interestingly, depleting NESPRIN2 led to an increase of H3K9me2 levels in the progenitor cells, indicating that forces applied on the nucleus are dependent on NESPRIN2<sup>43</sup> (Figures 6I and 6J). Thus, *in vitro*, similar to *in vivo*, the link between the nucleus via NESPRIN2 and the actin cytoskeleton is important for EC differentiation.

To compress the nucleus, we overlaid cells with an agarose pad at differentiation onset (D0), as previously used for other cell types.<sup>29,58</sup> After 6 days, we measured the height of the nuclei on confined and control cells stained with lamin B1 and observed a significant flattening (Figures 6K and 6L). We also counted the number of differentiating/differentiated cells and observed an in-

crease in their number following compression (Figures 6G and 6H). Thus, mechanical load on the cell is sufficient to deform the cell nucleus and induce ependymal differentiation. To evaluate whether mechanical loading compensated for deficiencies in the LINC complex and actin, we compressed cells depleted for *Arp3* or *Syne2* (Figures 6G and 6H). In both cases, we observed increased differentiation in depleted cells under compression compared with uncompressed cells. Taking the *in vitro* and *in vivo* experiments together, we conclude that nuclear deformation and EC differentiation go hand in hand, with nuclear deformation seemingly triggering EC differentiation.

## S-phase transition factors are downstream of nuclear deformations during EC differentiation

This result raises the question of what is transducing the mechanical deformation of the nucleus to the transcription factors that are known to control the differentiation of multiciliated cells.
<figure><img src="image_14.png" /><figcaption>**Figure 5.** NESPRIN2 localizes earlier during differentiation at the nuclear envelope than NESPRIN1 and promotes nuclear deformation and ependymal differentiation</figcaption></figure>

All staining were performed on whole mount lateral walls. (A) Staining of NESPRIN1 (gray, top) or NESPRIN2 (gray, bottom), and centrin2-GFP (Cen2GFP, green) during EC differentiation (P5). Small insets are 5× zoomed single-channel images corresponding to the orange dashed squares. Blue is Hoechst staining in the merge insets.

(B–F) Wild-type (B–D) or centrin2-GFP (E and F) mice transfected at P1 with mb-cherry as a reporter and either a control plasmid (Ctrl) or the shRNA against *Syne2* (to deplete NESPRIN2, sh*Syne2*).

(B) Staining of lamin B1 (gray) and FOP (green) during ependymal cell differentiation (P5). Transfected cells, either Ctrl (top row) or sh*Syne2* (bottom row), are circled with orange dashed lines (Transf). Top view is a maximum projection of z stacks. Side view is an Imaris 3D projection with segmented nuclei. The white dashed lines represent the plasma membrane.

(C and D) Quantification of the nuclear shape during the different steps of EC differentiation on Imaris segmented nuclei as in (B). (C) Pole-to-pole elongation index. (D) Distance between the center of the nucleus and the highest centriole. Error bars represent the SEM of *n* = 26/37 progenitors, 40/15 differentiating, and 31/30 multi-basal bodies (Multi BB) transfected cells (Ctrl/sh*Syne2*) from three mice; \* *p* ≤ 0.05; ns, *p* > 0.05; \*\* *p* ≤ 0.01 (Mann-Whitney). Control values are the same than in Figure 2 (experiments done at the same time).

(E) Staining of centrin2-GFP (Cen2GFP, green), mb-cherry (transfection reporter, red), and beta-catenin (βcat, blue) in differentiated cells (P15).

(F) Quantification of the percentage of transfected cells as in (E) remaining as progenitors (Prog), still differentiating (Diff), or differentiated (Multi). Error bars represent the SEM of six (Ctrl) or three (sh*Syne2*) experiments, *n* = 148 (Ctrl) and 84 (sh*Syne2*) transfected cells; The control values are the same than the one in Figure 4 (experiments performed at the same time). \*\*\* *p* ≤ 0.001 (two-way ANOVA followed by Sidak's multiple comparisons). Scale bar, 5 µm.

There is extensive evidence in other systems that the transcription factor Yes-associated protein (YAP) is involved in mechanotransduction.²⁷,⁵⁹ We therefore stained lateral ventricles at P5 with YAP/TAZ (Yes-associated protein/transcriptional coactivator with PDZ-binding motif) antibody and showed an increase of the YAP/TAZ levels in the cytoplasm, as well as a translocation of these proteins into the nucleus during ependymal differentiation (Figures S6A–S6C). However, mice mutant for YAP (YAP¹⁰⁾[lox/lox]; Nestin:Cre depleted from embryonic day 11.5 [E11.5]) still displayed ependymal markers (cilia and centrioles) at both P5 and P10, similar to control litter mates, suggesting that YAP is not required for triggering ependymal differentiation in the lateral ventricles (Figure S6D).

In searching for an alternative mechanotransduction pathway, we noticed that in other systems, mechanical strain or cell confinement triggers cell cycle events and notably the transition between G1 and S phases.²⁹,³⁰,³³ In this cycling cells, CDK2 is

known to phosphorylate RB1, triggering the release of E2F transcription factors from their interaction with RB1 and allowing them to activate S-phase-specific genes to transition out of G1.⁶⁰ In ECs, we know from previous work that CDK2 is also required for the early steps of multiciliated differentiation¹¹,⁶¹ and that RB1 is hyperphosphorylated in differentiating EC.⁶¹ Moreover, E2F4 and 5, along with the transcription regulators GEMC1 and MCIDAS, are required to activate transcription of specific genes for multiciliated cell differentiation.⁹ Finally, RBL2 (Retinoblastoma-Like Protein 2) in the airway inhibits differentiation through MCIDAS.⁶² In keeping with this, expression of a non-phosphorylable version of RB1⁶³ (hRB1ΔCDK) gave reduced differentiation compared with control cells (Figures 7A and 7B). This is unlikely to be due to a change in nuclear deformation, as H3K27me3 still increased during the differentiation (Figures S7A and S7B). Furthermore, we observed that MCIDAS overexpression did not induce rapid ependymal
<figure><img src="image_15.png" /></figure>

<figure><img src="image_16.png" /></figure>

<figure><img src="image_17.png" /></figure>

<figure><img src="image_18.png" /></figure>

<figure><img src="image_19.png" /></figure>

<figure><img src="image_20.png" /></figure>

<figure><img src="image_21.png" /></figure>

<figure><img src="image_22.png" /></figure>

<figure><img src="image_23.png" /></figure>

<figure><img src="image_24.png" /><figcaption>**Figure 6.** Confining the progenitor cells in vitro induces ependymal differentiation and rescues the absence of NESPRIN2</figcaption></figure>

All staining were performed during EC differentiation in vitro 6 days after serum withdrawal (D6). (A) Staining of lamina B1 (gray), Sas6 (red), and centrioles (Cen2GFP, green). Top (top) and side (bottom) views are Imaris 3D projection of a z stack.

(B and C) Quantification of nuclear shapes on Imaris segmented nuclei. (B) Volume. (C) Pole-to-pole elongation index. Error bars represent the SEM of *n* = 174 progenitors (Prog), 60 differentiating (Diff), and 67 multi-basal body (Multi) cells from five different cultures. \*p ≤ 0.05; \*\*p ≤ 0.001 (one-way ANOVA followed by Dunn's multiple comparison).

(D) Staining of H3K27me3 (gray, top) or H3K9me2 (gray, bottom) and FOP (centrioles, green).

(E and F) Quantification of the fluorescence intensity of H3K27me3 (E) or H3K9me2 (F) staining at the nuclear envelope at different stages of differentiation (Prog, progenitors; Diff, differentiating; Multi, multiciliated differentiated cells) on images as in (D) normalized to the mean of intensity in progenitors of the same image. Error bar represent the SEM of *n* = 407, 111, and 275 cells for Prog, Diff, and Multi cells, respectively, in H3K27me3 staining and *n* = 681, 143, and 338 cells for Prog, Diff, and Multi, respectively, in H3K9me2 staining in three independent experiments each. \*\*p ≤ 0.001; \*\*p < 0.01 (one-way ANOVA followed by Dunn's multiple comparison).

(G and H) Progenitor cells in vitro were transfected with shRNA either control (Ctrl) or against Arp3 (shArp3) or against Syne2 (shSyne2) at D0. They were then confined (Pad) or not (No Pad) under an agarose pad for 6 days. (G) Staining of transfected control or confined cells with FOP (green), Foxj1 (transcription factor specific of multiciliated differentiating and differentiated cells, red), and Hoechst (DNA, blue). Transfected cells were circled with orange dashed line (transfection). (H) Quantification of the percentage of differentiating (Diff) + differentiated (Multi) transfected cells. Error bars represent the SEM of at least six coverslips

(over three independent experiments) and over 115 transfected cells. \*\*\*p ≤ 0.001 (Mann-Whitney).

(I and J) Cells were transfected at D0 with Tomato as a reporter and either a control plasmid (Ctrl) or an shRNA against Syne2 (shSyne2). (I) Staining of FOP (green) and H3K9me2 (gray) in cells either control (Ctrl) or depleted for NESPRIN2 (shSyne2). (J) Quantification of the levels of H3K9me2 fluorescence intensity in Ctrl or shSyne2 cells. Error bar represent the SEM of *n* = 47 or 49 cells for Ctrl and shSyne2, respectively, in two independent experiments. \*\*\*p ≤ 0.0001 (Mann-Whitney).

(K and L) Progenitor cells in vitro were confined (Pad) or not (No Pad) under an agarose pad from D0 for 6 days. (K) 3D Imaris view from the side of the cells. (L) Quantification of the nucleus thickness of progenitor cells with Imaris software on image as in (K). Error bars represent the SEM of 156/165 nuclei measured on three independent experiments. \*\*\*p ≤ 0.001 (Mann-Whitney). Scale bar, 5 µm.

differentiation in cells expressing non-phosphorylable RB1 (Figures 7C, 7D, and S7C), suggesting that RB1 phosphorylation/release of E2F is important for MCIDAS activation during differentiation. Altogether, these results suggest that the phosphorylation of RB1 is required for ependymal differentiation through MCIDAS activation.

A possibility, therefore, is that nuclear deformation during differentiation leads to the phosphorylation/degradation of RB1 releasing E2F to activate MCIDAS. To test this hypothesis, we depleted Arp3 in cells of the lateral wall (from differentiation onset) a treatment that gave impaired nuclear deformation

and ependymal differentiation (Figure 2). We observed that under these conditions phospho-RB1 failed to accumulate in the nucleus of differentiating cells compared with control (Figures 7E and 7F). Conversely, we rescued differentiation in Arp3 depletion conditions by co-depleting Rb1 (Figures 7G and 7H) with previously validated shRNA⁶⁴ (Figures 7E and 7F). Note that Rb1 depletion had no effect on differentiation on its own (Figures S7D and S7E).

Putting all of these data together, we conclude that RB1 phosphorylation is a likely player in the pathway connecting actin accumulation and nuclear deformation to changes in differentiation.
<figure><img src="image_25.png" /></figure>

differentiated cells (P15) of wild-type mice transfected with a control plasmid (Ctrl = shNeg-IRES-GFP+ shNeg) or shArp3 (shArp3-IRES-GFP+ shNeg) or both shArp3 and shRb1 (shArp3-IRES-GFP+ shRb1). IRES containing constructs were used less concentrated than the other transfection reporter (potentially explaining the decreased effect of shArp3 compared with Figure 2).

(H) Quantification of the percentage of transfected cells as in (G), remaining as progenitors (Prog), still differentiating (Diff), or differentiated in ECs (Multi). Error bars represent the SEM of six (Ctrl) or six (shArp3) or three (shArp3 + shRb1) experiments, *n* = 109 (Ctrl), 155 (shArp3), and 68 (shArp3 + shRb1) transfected cells; \*\*\*p ≤ 0.001 (two-way ANOVA followed by Sidak's multiple comparisons); \*p ≤ 0.05. Scale bar, 5 µm.

DISCUSSION

Impairing nuclear deformation either by decreasing actin (shArp3, Figure 2) or by severing the link between the nucleus and the actin cytoskeleton (KASH expression or shSyne2, Figures 4 and 5), impaired EC differentiation. Conversely, forcing nuclear deformation by increasing actin polymerization (Arpin<sup>KO/lox</sup>; Cre or shSyne1) (Figures 3, S3, and S5) or compressing the cells (Figure 6) accelerated differentiation. Our results thus show that mechanical constraints on the nucleus are important for EC differentiation in the mouse brain. In line with the presence of mechanical forces on the nucleus, histone H3

<caption>**Figure 7. RB1 is involved in ependymal dif- ferentiation following nuclear deformation**</caption>

All staining were performed on whole mount lateral walls. (A) Staining of centrin2-GFP (Cen2GFP, green), mb-cherry (transfection reporter, red), and beta-catenin (βcat, blue) in differentiated cells (P15) on centrin2-GFP mice transfected at P1 with mb-cherry and either a control plasmid (Ctrl) or a plasmid coding for an unphosphorylable RB1 (hRB1-ΔCDK). (B) Quantification of the percentage of transfected cells as in (A) remaining as progenitors (Prog), still differentiating (Diff), or differentiated (Multi). Error bars represent the SEM of four experiments, *n* = 146 (Ctrl) and 148 (hRB1-ΔCDK) transfected cells; \*\*\*p ≤ 0.001 (two-way ANOVA followed by Sidak's multiple comparisons).

(C) Staining of FOP (green), GFP (transfection reporter, red), and beta-catenin (βcat, blue) in early differentiating cells (P3) of wild-type mice transfected at P1 with control plasmids (Ctrl = shNeg-IRES-GFP+ shNeg) or MCIDAS (shNeg-IRES-GFP+ MCIDAS) or both MCIDAS and unphosphorylatable RB1 (hRB1-ΔCDK-IRES-GFP+ MCIDAS).

(D) Quantification of the percentage of transfected cells as in (C) in the different stages of differentiation: progenitors (Prog), differentiating (Diff), or differentiated (Multi). Error bars represent the SEM of three (Ctrl) or eight (MCIDAS) or five (MCIDAS + hRB1-ΔCDK) experiments, *n* = 75 (Ctrl), 198 (MCIDAS), and 123 (MCIDAS + hRB1-ΔCDK) transfected cells; \*\*\*p ≤ 0.0001; \*\*p ≤ 0.01; \*p ≤ 0.05 (two-way ANOVA followed by Sidak's multiple comparisons).

(E) Staining of phospho-RB1 (green) and FOP (red) in differentiating cells transfected with mb-cherry (Transf, orange dashed circle) and either a control plasmid (Ctrl, top row), an shRNA against Arp3 (shArp3) or an shRNA against Rb1 (shRb1).

(F) Quantification of the fluorescence intensity of nuclear phospho-RB1 of transfected cells as in (D) normalized to the mean intensity of nuclear phospho-RB1 in three surrounding untransfected progenitors. Error bars represent the SEM of *n* = 19/16/16 (Ctrl/shArp3/shRb1) transfected cells. \*\*\*p ≤ 0.001 (Mann-Whitney).

(G) Staining of FOP (green), GFP (transfection reporter, red), and beta-catenin (βcat, blue) in

methylation (Figures 1E–1G and 6D–6F) and DNA damage<sup>61</sup> are observed during differentiation, similar to reports in other cell types submitted to mechanical stress.<sup>35,43</sup> Interestingly, compression of the nucleus rescued the effect of *Syne2* depletion on differentiation to the levels of control without compression, whereas it only partly re-established differentiation in cells depleted for *Arp3* (Figures 6G and 6H). This is consistent with actin playing a role even when not linked through the LINC complex to the nucleus.

Independent of the Hippo pathway, it was shown that YAP entry in the nucleus is sensitive to forces applied on the nucleus through the opening of nuclear pores.<sup>27</sup> Interestingly, the YAP
pathway is also involved in cell proliferation and differentiation in
several tissues.⁵⁹ In the lung, depletion of YAP during cell spec-
ification prevents the formation of multiciliated cells at the
expense of progenitor and goblet cells. However, at differentia-
tion onset, expression of a nuclear YAP also prevents lung multi-
ciliated cell differentiation.⁶⁵,⁶⁶ Similarly, in the brain, YAP is
involved in the specification/integrity of ependymal progenitor
cells in the aqueduct and, to a lesser extent, in the cortex.⁶⁷
Consequently, YAP absence leads to the blockage of the rostral
aqueduct and non-communicating hydrocephalus. However,
removing YAP does not prevent ependymal differentiation
(Figure S6D).

In cycling cells, CDK2 is known to phosphorylate RB1, triggering the release of E2F transcription factors from their interaction with RB1 and allowing them to activate S-phase-specific genes to transition out of G1.⁶⁰ Interestingly, mechanical strain or cell confinement also triggers cell cycle events and notably the transition between G1 and S phases.²⁹,³⁰,³³ Whether this depends on RB1 phosphorylation was not assessed. Multiciliated cell differentiation depends on the transcription regulators GEMC1 and MCIDAS that together with E2F4 or 5 activate transcription of specific genes.⁹ It also depends on CDK2,¹¹ and RB1 is hyperphosphorylated,⁶¹ even if the cells are post mitotic. We show here that RB1 phosphorylation is important for ependymal differentiation in a pathway involving MCIDAS (Figures 7A-7D) and propose that RB1 phosphorylation could provide a possible explanation for how nuclear deformation impacts transcription factor activity for differentiation. Indeed, removal of RB1 rescued ependymal differentiation in the absence of nuclear deformation (shArp3, Figures 7G and 7H). Moreover, RB1 phosphorylation depends on nuclear deformation (Figures 7E and 7F). Thus, nuclear deformation may induce RB1 phosphorylation that will allow the release of E2F present in the cells and the activation of the multiciliated transcriptional regulators. Cells will remain in undifferentiated state until the nucleus is constrained by the actin cytoskeleton. This constraint will then induce the first steps of cell-cycle-like activation that will trigger ependymal differentiation without inducing DNA replication.⁶¹

At this stage, we cannot rule out other mechanisms linking nu-
clear deformation to differentiation. It is, for example, unclear
whether the changes in histone H3 methylation patterns (going
from H3K9me2 to H3K27me3, Figures 1E–1G and 6D–6F), which
depends on the LINC complex (Figures 6I and 6J), are neces-
sary/sufficient for the differentiation. Interestingly, RB1 interacts
with HP1/SUV39H1 that are essential for H3K9 methylation.⁶⁸
Thus, RB1 phosphorylation might influence H3K9 methylation.
Similarly, nuclear pore or nuclear channel opening following nu-
clear deformation may play a role in the differentiation,⁶⁹ even if it
does not imply YAP in our model (Figure S6).

The mechanical cues leading to actin accumulation that will
act on the nucleus are not known. Neural stem cell progenitors
are subjected to a variety of forces ranging from tissue growth
(not accounted by division⁷), changes in morphology (going
from elongated to more cuboidal during differentiation),
and pressure or shear stress generated by the cerebrospinal
fluid.¹²,⁷⁰ Interestingly, stretch-activated channels, such as
PIEZO1 (mechanosensitive ion channel), are involved in neural
stem cell differentiation⁷¹ and may contribute to some aspect
of multiciliated cell differentiation.⁷²

Limitations of the study

This study demonstrates that nuclear deformation by the actin cytoskeleton triggers RB1 phosphorylation and activation of MCIDAS, a transcriptional regulator of multiciliated cell. However, the specific mechanisms by which RB1 is phosphorylated following deformation remain to be addressed.

RESOURCE AVAILABILITY

Lead contact

Nathalie Delgehyr: delgehyr@bio.ens.psl.eu.

Materials availability

All unique/stable reagents generated in this study are available from the lead contact.

Data and code availability

The data that support the findings of this study are available within the article and supplemental information or are available from the authors upon request.

ACKNOWLEDGMENTS

We are grateful to J. Gleeson for providing the centrin2-GFP mice. We thank
X. Morin for the CAAGS-mb-cherry and CAAGS-Cre plasmids, I. Caillé
and S. Scotto for the shRNA backbones, A. Merdes for the KASH plasmid,
S. Dowdy for the hRB1ΔCDK plasmid, G. Gundersen for the NESPRIN2
antibody, S. Taraviras for the MCIDAS plasmid, N.J. Quintyne for the
p150-CC1 plasmid, and M. Pende and E.N. Olson for the Yap<sup>lox</sup> mice. We
are grateful to A. Delecourt, E. Touzalin, G. Firmin, and L. Le Moal for their
help with the animal husbandry. We thank M. Bornens for all his input
over the year. We are grateful to all members of the Spassky laboratory.
The team was funded by the Agence Nationale de la Recherche Investisse-
ments d'avenir (ANR-10-LABX-54MEMO LIFE, ANR-11-IDEX-0001-02 PSL*
Research University, ANR-10-IDEX-0001-02 PSL, and ANR-10-LABX-31).
The Spassky team is supported by the Inserm, the CNRS, the ENS, the
ANR (ANR-20-CE45-0019, ANR-21-CE16-0016, and ANR-22-CE16-0011),
the Fondation pour la Recherche Médicale (FRMEQU202103012767), and
the Fondation Pierre-Gilles de Gennes (FPGG03).

AUTHOR CONTRIBUTIONS

Investigation and formal analysis, M.B., A. Mahuzier, S.K.A., A. Marty, A.S., M.K.D., and N.D.; conceptualization, M.B., A. Mahuzier, and N.D.; methodology and resources, A.M.L.D., M.F., and A.B.; writing and supervision, J.P., N.S., A.Y., and N.D.; funding acquisition, A. Meunier, A.Y., J.P., N.S., and N.D.

DECLARATION OF INTERESTS

The authors declare no competing interests.

STAR★METHODS

Detailed methods are provided in the online version of this paper and include
the following:

● KEY RESOURCES TABLE

● EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

○ Transgenic mice

○ Cell cultures

○ Bacterial strain

● METHOD DETAILS

○ Plasmids

○ Postnatal electroporation

○ Immunostaining

○ Microscopy

○ Drug treatments

○ Agarose pad
* *In vitro* transfection

* 3D modelling

* QUANTIFICATION AND STATISTICAL ANALYSIS

* Quantification

* Statistics

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.devcel.2024.11.008.

Received: October 2, 2023

Revised: July 16, 2024

Accepted: November 8, 2024

Published: December 10, 2025

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Antibodies</strong></td></tr><tr><td>Rabbit anti-Living Colors DsRed</td><td>Clontech</td><td>Cat#632496; RRID: AB_10013483</td></tr><tr><td>Rat anti-RFP</td><td>Chromotek</td><td>Cat#5F8-100; RRID: AB_2336064</td></tr><tr><td>Chicken anti-GFP</td><td>Aves</td><td>Cat#GFP1020; RRID: AB_10000240</td></tr><tr><td>mouse IgG2b anti-FGFR10P</td><td>abnova</td><td>Cat#H00011116-MO; RRID: AB_538806</td></tr><tr><td>mouse IgG1 anti-beta-catenin</td><td>Milipore</td><td>Cat#05-665; RRID: AB_309887</td></tr><tr><td>mouse IgG2b anti-Sas6</td><td>Santa-Cruz</td><td>Cat#Sc-81431; RRID: AB_1128357</td></tr><tr><td>mouse IgG1 anti-LaminB1</td><td>Santa-Cruz</td><td>Cat#Sc-374015; RRID: AB_10947408</td></tr><tr><td>Rabbit anti-Deup1</td><td>Our lab</td><td>Mercey et al.<sup>73</sup></td></tr><tr><td>Rabbit anti-phospho S807/811-RB</td><td>Cell Signaling</td><td>Cat#8516; RRID: AB_11178658</td></tr><tr><td>mouse IgG1 anti-Foxj1 (2A5)</td><td>Thermo-Fisher</td><td>Cat#14-9965-82; RRID: AB_1548835</td></tr><tr><td>rabbit anti-Nesprin2</td><td>Greg Gundersen</td><td>Luxton et al.<sup>74</sup></td></tr><tr><td>rat anti-tubulin YL1/2</td><td>Sigma</td><td>Cat#MAB1864; RRID: AB_2210391</td></tr><tr><td>mouse IgG2a anti-centrin (20H5)</td><td>Sigma</td><td>Cat#04-1624; RRID: AB_10563501</td></tr><tr><td>mouse IgG1 anti-Nesprin-1</td><td>Hybridoma bank</td><td>Cat#MANNES1A; RRID: AB_2753299</td></tr><tr><td>mouse IgG2a anti-H3K9me2</td><td>abcam</td><td>Cat#ab1220; RRID: AB_449854</td></tr><tr><td>rabbit anti-H3K27me3</td><td>Cell Signaling</td><td>Cat#9733S; RRID: AB_2616029</td></tr><tr><td>rabbit anti-YAP/TAZ (D24E4)</td><td>Cell Signaling</td><td>Cat#8418; RRID: AB_10950494</td></tr><tr><td>mouse IgG1 polyglutamylation modification (GT335)</td><td>Adipogen</td><td>Cat#AG-20B-0020; RRID: AB_2490210</td></tr><tr><td colspan="3"><strong>Bacterial and virus strains</strong></td></tr><tr><td>pCMV HA hRB1 delta CDK</td><td>Addgene</td><td>Cat# 58906; Narasimha et al.<sup>63</sup></td></tr><tr><td colspan="3"><strong>Experimental models: Organisms/strains</strong></td></tr><tr><td>RjOrl:SWISS</td><td>Janvier labs</td><td>N/A</td></tr><tr><td>B6D2-Tg(CAG-EGFP/CETN2)3-4Jgg/J</td><td>The Jackson Laboratory</td><td>Cat# 008234; Higginbotham et al.<sup>38</sup></td></tr><tr><td>C57BL/6-YAPdKO</td><td>Eric Olson</td><td>Xin et al.<sup>75</sup></td></tr><tr><td>B6D2-ArpindKO</td><td>Ana-Maria Lennon-Duménil</td><td>Alraies et al.<sup>49</sup></td></tr><tr><td>C57BL/6-Tg(Nestin-Cre)</td><td>Johan Dahlstrand</td><td>Dahlstrand et al.<sup>76</sup></td></tr><tr><td colspan="3"><strong>Oligonucleotides</strong></td></tr><tr><td>shArp3: TTAGCTCTCTTCTACATCTGC</td><td>-</td><td>Drummond et al.<sup>48</sup></td></tr><tr><td>shSyne2: AGCCACAGAACTCCAAAAT</td><td>-</td><td>Espigat-Georger et al.<sup>57</sup></td></tr><tr><td>hSyne1: GCTCCTGCTGCTGCTTATT</td><td>-</td><td>Espigat-Georger et al.<sup>57</sup></td></tr><tr><td>shRb1: AGTTAGGACTGTTATGAAT</td><td>-</td><td>Taura et al.<sup>64</sup></td></tr><tr><td colspan="3"><strong>Recombinant DNA</strong></td></tr><tr><td>pCAGGS Membrane-Cherry</td><td>Xavier Morin</td><td>Peyre et al.<sup>77</sup></td></tr><tr><td>pcDNA-U6-shRNA</td><td>Isabelle Caillé; Sophie Scotto</td><td>Scotto-Lomassese et al.<sup>78</sup></td></tr><tr><td>pTRIP-U6-shRNA-IRES-PGK-GFP-WPRE</td><td>Isabelle Caillé; Sophie Scotto</td><td>Scotto-Lomassese et al.<sup>78</sup></td></tr><tr><td>pCAGGS-Cre</td><td>Xavier Morin</td><td>Morin et al.<sup>79</sup></td></tr><tr><td>pdsRed-p150Glued-CC1</td><td>NJ Quintyne</td><td>Quintyne et al.<sup>80</sup></td></tr><tr><td>pEGFP-C2-KASH</td><td>Andreas Merdes</td><td>Espigat-Georger et al.<sup>57</sup></td></tr><tr><td>MCIDAS-GFP</td><td>S Taraviras</td><td>Kyrousi et al.<sup>81</sup></td></tr><tr><td>pCMV-tdTomato</td><td>Clontech</td><td>Cat#632534</td></tr><tr><td>pIRES2-EGFP</td><td>Clontech</td><td>Cat#6029</td></tr><tr><td colspan="3"><strong>Software and algorithms</strong></td></tr><tr><td>ImageJ</td><td>Wayne Rasband</td><td>https://imagej.net/ij/</td></tr><tr><td>Imaris9.8.0</td><td>Oxford Instrument</td><td>https://imaris.oxinst.com/</td></tr></tbody></table>
## EXPERIMENTAL MODEL AND STUDY PARTICIPANT DETAILS

### Transgenic mice

All animal studies were performed in accordance with the guidelines of the European Community and French Ministry of Agriculture and were approved by the Ethic comity Charles Darwin (C2EA-05) and "Direction départementale de la protection des populations de Paris", (Approval number Ce5/2012/107; APAFiS #9343). Both male and female animals were used in the study and no differences between genders were observed. Specific-pathogen free mice were housed under standard conditions (12 h light/dark cycle; lights on at 7:00 A.M.) in the conventional animal facility of IBENS. Unlimited food and water supply were provided. Animals were electroporated at Post-natal day 1 (P1) and used at P3, P5 or P15 depending on the experiments. Most of the mouse strains have already been described. Wild-type are RjOrl:SWISS (Janvier labs). Cen2GFP³⁸ (B6D2-Tg(CAG-EGFP/CETN2)3-4Jgg/J, The Jackson Laboratory) were used heterozygous for the transgene. B6D2-Arpin conditional knockout mice (Arpin<sup>lox</sup>) were generated at CIPHE (Centre d'Immunophénomique, Marseille, France) using CRISPR cas-9 technology,⁴⁹ genotypes are indicated in the corresponding Figure legends. C57BL/6-YAP conditional knockout (Yap<sup>lox/lox</sup>)⁷⁵ were crossed with C57BL/6-Tg(Nestin-Cre)⁷⁶; Yap<sup>lox/+</sup>, genotypes are indicated in the corresponding Figure legends.

### Cell cultures

Differentiating ependymal cells were cultured as previously described.⁵⁴ Brains were removed from the animal and dissected in cold PBS. Hypothalamus, meninges, bulbs and choroid plexus were removed. What remains was cut in small pieces, incubated in the culture incubator in 1ml DMEM/ Glutamax containing 1% Penicillin/ Streptomycine (PS, 10,000 Units; Gibco), 30 µl of Papain (stock 2x crystalline suspension, Worthington), 15 µl of DNAse I (stock 1% in water; Worthington), 24 µl of L-Cystein (stock 12 mg/ml in water; Sigma) per brain for 45 minutes. The brain pieces were then washed with L15 buffer containing 1mg/ml of trypsin inhibitor and 200µl of DNAse I, followed by a wash with L15. They were then dissociated manually using a 1ml tip (maximum 10 back and forth in the tip) and centrifuge to be plated in DMEM/ Glutamax containing 1% PS and 10% Fetal Bovine Serum (FBS) on 25cm² flask coated with poly-L-Lysin. The cells were expended during 5 days and then passage to be inoculated on glass coverslips, coated with poly-L-Lysin, at 0.2x10⁶ cells per 20µl drop. After an hour, 500 µl of complete media was added (DMEM/ Glutamax containing 1% PS and 10% Fetal Bovine Serum (FBS)) on the cells. The day after cells were rinsed three times in DMEM/ Glutamax containing 1% PS without FBS. This is considered at Day 0 for differentiation.

### Bacterial strain

E. coli DH5α were used for the cloning and expression of plasmids. Cells were cultured in LB medium supplemented with necessary antibiotics.

## METHOD DETAILS

### Plasmids

We cloned the shRNA sequences in the shRNA backbone vectors pcDNA-U6-shRNA and pTRIP-U6-shRNA-IRES-PGK-GFP-WPRE as previously described.⁷⁸ The control shRNA (shCtrl) was generated from AllStars Negative Control siRNA (Qiagen), which does not recognize mammalian RNAs. The reporter plasmid encoding membrane Cherry (mb-cherry) under the control of the CAGGS promoter⁷⁷ was co-transfected with pcDNA-U6-shRNA and was a gift from Xavier Morin. All cells expressing the shRNA-GFP (pTRIP-U6-shRNA-IRES-PGK-GFP-WPRE) or cells expressing pCAGGS-mbCherry (mb-cherry), which were co-transfected with the untagged pcDNA-U6-shRNA, were considered to be depleted.

shRNA sequences were validated in referenced paper and in this study: shArp3⁴⁸: TTAGCTCTCTTCTACATCTGC (Figures S3A and S3B); shSyne2⁵⁷: AGCCACAGAACTCCAAAAT (Figures S4H and S4I); shSyne1⁵⁷: GCTCCTGCTGCTGCTTATT (Figures S4E and S4G); shRb1⁶⁴: AGTTAGGACTGTTATGAAT (Figures 7E and 7F).

The pCAGGS-Cre⁷⁹ plasmid is gift from X. Morin. The KASH domain construct⁵⁷ is a gift from A. Merdes and is tagged with GFP. The p150Glued-CC1 domain construct⁵⁰ is a gift from NJ. Quintyne and is tagged with dsRed. The pCMV HA hRB1 delta CDK⁶³ was a gift from S. Dowdy (Addgene plasmid # 58906; http://n2t.net/addgene:58906; RRID: Addgene_58906). HA hRB1 delta CDK was subcloned at BamHI restriction site on pIRES2-EGFP vector (Clontech). The MCIDAS-GFP construct⁸¹ was a gift from S. Taraviras, in our hand the GFP was barely detectable. pCMV-tdTomato is from Clontech.

### Postnatal electroporation

Electroporation was performed on one-day-old pups anaesthetized 1 minute on ice as previously described.⁸² An elongated glass capillary filled with the 2 µl DNA solution diluted in PBS with 0.25% Fast-Green was inserted in the brain ventricle. With a syringe, the solution was injected and could be seen filling the ventricles. Animals were then subjected to five electrical pulses (100 Volts, 99 ms length, separated by 90 ms intervals) using the CUY21 edit device (Nepagene, Chiba, Japan) and 10 mm tweezer electrodes (CUY650P10, Nepagene) coated with conductive gel (Parker laboratory). Electroporated animals were reanimated for several minutes on a 37°C heating plate before being returned to the mother. shRNA in the U6 backbone (untagged) (2.5 µg) were co-transfected with the reporter plasmid mb-cherry or with plasmids encoding shRNA in the pTRIP backbone (IRES GFP) (1.5 µg).
MCIDAS-GFP (2 µg) was electroporated either with pTRIP-shNeg or with HA hRB1 delta CDK-IRES2GFP (2 µg). All cells expressing the reporter were assumed to express both the reporter and the plasmid of interest.

## Immunostaining

Lateral walls of the lateral brain ventricles were dissected, fixed, and immunostained as previously described.⁵⁴ Brains were removed from the animal and dissected in cold PBS. Hypothalamus, medial and dorsal walls were removed to make the lateral wall accessible. The same area (rostro-dorsal) of the lateral walls was used throughout the study. The tissue was treated with 0.1% triton in BRB80 (80 mM K-Pipes pH6.8; 1 mM MgCl2; 1 mM Na-EGTA) for 1 min and fixed in methanol at -20°C for 10 min, most of the time. For phalloidin or YAP/TAZ stained tissues, they were detergent-treated and then fixed in 4% PFA in BRB80 for 8 min, for phospho-RB1 and H3K37me3 staining or tissue electroporated with an IRES-GFP containing constructs, the tissue were fixed in 4% PFA in BRB80 for 20 minutes. The following antibodies were used: rabbit or rat anti-dsRed (1:500, Clontech 632496 or 5F8 1:400, Chromotek 5f8-100); chicken anti-GFP (1:1600, Aves GFP-1020); mouse IgG2b anti-FOP (FGFR10P, 1:1000, Abnova H00011116-MO), mouse IgG1 anti-beta-catenin (1:1000, Millipore05-665), mouse IgG2b anti-Sas6 (1:700, Santa-Cruz Sc-81431), mouse IgG1 anti-LaminB1 (1:1000, Santa-Cruz Sc-374015), Rabbit anti-Deup1 (1:2000),⁷³ Rabbit anti-phospho S807/811-RB (1:1000, Cell Signaling 8516), mouse IgG1 anti-Foxj1 (2A5, 1:1000, Thermo Fisher 14-9965-82), rabbit anti-Nesprin2 (1/100, gift from Greg Gundersen)⁷⁴, rat anti-tubulin YL1/2 (1:100, Sigma MAB1864), mouse IgG2a anti-Centrin (20H5 1:1000, Sigma 04-1624), mouse IgG1 anti-Nesprin-1 (1:50, Hybridoma bank MANES1A), mouse IgG2a anti-H3K9me2 (1:200, abcam ab1220), rabbit anti-H3K27me3 (1:100, Cell Signaling 9733S), rabbit anti-YAP/TAZ (1:50, D24E4 Cell Signaling 8418), mouse IgG1 polyglutamylation modification (GT335, 1:1000, Adipogen AG-20B-0020), and species-specific Alexa Fluor secondary antibodies (1:800, Invitrogen). Phalloidin-A488 or A568 were used at a dilution of 1:50 with primary and secondary antibodies. Primary antibodies were incubated overnight at 4°C or 3h at room temperature in 0.1% triton/10%FBS/PBS. Washed 2x 10 minutes in 0.1%triton/PBS before to proceed with the secondary antibodies for 1h in 0.1% triton/10%FBS/PBS and similar washes. Tissue were then further dissected to keep mainly the lateral wall and flat mounted in Fluoromount.

## Microscopy

### Optical imagery

Images (230-nm z-steps) of fixed samples were obtained with an inverted LSM 880 Airyscan (Zeiss) with a 63x 1.4NA oil-immersion objectives on confocal mode, except for the phalloidin staining where it was used in the airyscan mode (180nm z-steps), and Zen2 software. Z-stack projections are shown. Only the 7 most apical planes are shown for phalloidin staining. For P150-CC1 and KASH nuclei some intermediate Z were removed to allow visualization of the transfected nuclei in the depth of the tissue. The KASH-GFP around the nucleus is not visible on images representative of EC differentiation as the Z-stack including the apical surface does not include the whole nuclei deeper in the tissue.

### Drug treatments

Lateral wall explants were immersed for the indicated times at 37°C with 5% CO₂ in DMEM-Glutamax supplemented with 10% FBS, 1% PS and cytochalasin-D (2µM, Sigma) to depolymerize actin, SMIFH2 (60µM, Sigma) to inhibit formin,⁸³ CK666 (100µM, Sigma) to inhibit the Arp2/3 complex, Blebbistatin (100µM, Sigma) or Nocodazole (10µM, Sigma) to depolymerize microtubules. The lateral ventricular wall of the opposite side of each mouse brain were used as control and immersed in the same media containing an amount of DMSO equivalent to that of the drug.

### Agarose pad

To compress the nuclei, we prepared 5mL of 2% ultra-pure agarose (FMC Bio-Products) in serum-free DMEM- Glutamax medium in a 60-mm Petri-dish. We made sure that the plates were on a flat surface before to pour the agarose. A 33-mm diameter agarose pad was cut out and deposited gently on cells grown on coverslips immerged in 3ml of DMEM/ Glutamax containing 1% PS in a 6-well plate. A 32-mm glass coverslip surmounted of a 540 mg washer was carefully added above to maintain the cushion for compression.²⁹

### In vitro transfection

We transfected 10ng of the plasmid of interest with 10ng of reporter (pCMV-tdTomato) on 0.2x10⁶ cells in suspension (before D0) with 0.04 µL of Jetprime (Polyplus) according to manufacturer protocol. The cells were seeded onto a drop of 20µL on a coverslip coated with poly-lysine. 500 µL of DMEM-Glutamax complemented with 10% FBS and 1% PS was added after 1 hour of incubation. Media was changed to DMEM-Glutamax 1% PS without serum the day after (D0). Cells were fixed and analyzed 5 or 6 days after serum starvation (D5 or D6). Cells expressing the reporter were considered as co-transfected.

## 3D modelling

Imaris 9.8.0 software was used for 3D modelling.

All results were obtained in at least three independent experiments on at least three different animals or cell cultures. The numbers of biological replicates (n) are indicated in each figure legends. The experiments were not randomized and no statistical methods were used to predetermine sample size.
# QUANTIFICATION AND STATISTICAL ANALYSIS

## Quantification

All analyses were performed on images of the rostro-dorsal part of the lateral walls of the lateral ventricles to maximize reproducibility.

## Measurement of phalloidin or YL1/2 fluorescence intensity

Total mean actin (phalloidin fluorescence) or Tyr-tubulin (YL1/2 staining) intensity of fluorescence within cell borders were measured with ImageJ software on the sum projection of the 7 most apical z-stack sections of the cells. Quantifications were normalised with respect to the mean signal intensity in the three closest non-transfected progenitor cells or the mean intensity of progenitor cells of the same image. The results are expressed as the ratio of the values of the cells over the mean values of the progenitor cells.

## Characterisation of nuclear shape

Nuclei of cells co-transfected with mb-cherry were acquired for their Lamin-B1 staining. Analyses were performed with the Imaris software. For *in vivo* samples, a first loose manual segmentation was performed to exclude nuclei of the un-transfected cells. This step was not required for cells *in vitro*. Then an automatic segmentation was performed using Surface and 0.2 grain size resolution. The volume is a quantification of how much space the Surface object occupies (Imaris reference manual). To obtain the pole to pole elongation index, the nucleus is fitted to an ellipse, the dimension of the three principal axes (a, b, c) of the best-fitted ellipsoid into the surface, the moments of inertia along the axes of ellipsoid are computed and optimized to match the moment of inertia of the surface. The ellipsoid has homogeneous mass distribution within the surface object. The pole to pole elongation index is the ellipsoid prolate; define as $e_{prolate} = (2a^2/a^2+b^2)^*(1-((a^2+b^2)/2c^2))$ (Imaris reference manual).

The Centrin or FOP staining were segmented using dot and 0.25 µm size resolution. The distance of the nucleus to the surface was calculated as the difference in z position of the highest centriole (anchored to the plasma membrane with primary or motile cilia) and the centre of the nucleus. Centre and not the highest part of the nucleus were taken into account, as they are more representative of the nucleus position in a very crowded environment.

## Characterisation of cell differentiation stages

Only transfected cells with an apical contact at the surface of the ventricle were taken in account. When cells were stained with FOP or Centrin2-GFP, progenitors were considered to have only 2 dots, differentiating cells unresolved or donut like structures, multici- liated cells several resolved dots.

Sas6 or Deup1 staining, respectively pro-centrioles or deuterosomes, were used to confirm the differentiating cells when possible with the antibodies combinations and fixations.

To increase reproducibility, we counted the stages of the cells surrounding the transfected cells. At P15, images with 60 to 80% (average 65.8±1.7sem) multiciliated cells were taken in account. At P5, images with 60 to 70% (average 65.6% ±0.4sem) progen- itors cells were taken in account.

## Measurement of H3K27me3 and H3K9me2 levels in vivo

Total mean of H3K27me3 or H3K9me2 intensity of fluorescence were measured with ImageJ software on the median plane of the nucleus of cells expressing membrane-Cherry. The measurements were normalized with respect to the mean signal intensity in three closest un-transfected progenitor cells.

## Measurement of H3K27me3, H3K9me2 and NESPRIN2 levels in culture

Total mean of H3K27me3, H3K9me2 or NESPRIN2 intensity of fluorescence were measured with ImageJ software in differentiating ependymal cells *in vitro* on maximum intensity projection of the cell nuclei. Segmentations of the nuclei were performed using auto- matic thresholding with a triangle filter on Hoechst staining. Background taken in three independent zones in the images were sub- tracted from the mean value of NESPRIN2 levels for all measurements. The quantification was normalised with respect to the mean signal intensity in the non-transfected progenitor cells or the mean intensity of progenitor cells of the same image. The results are expressed as the ratio of the values of the cells over the mean values of the progenitor cells.

## Measurement of NESPRIN1 levels in culture

NESPRIN1 is localized both at the centrosome and at the nuclear envelope. At the centrosome, NESPRIN1 levels were quantified with ImageJ software by using a circle of 3.8 µm² centred on the centrosome on maximum intensity projection images. To account for background, the minimum intensity pixel in this area was subtracted from the mean. The levels were normalized with the mean intensity at the centrosome of un-transfected progenitor cells in the same image. For nuclear quantification, as centrosome were often located on top of the nucleus, we decided to quantify the nuclear envelope on the nucleus middle plan with ImageJ software by outlining it with a 2 pixel size line. Minimal pixel was subtracted from the mean to take in account the background. Intensity were normalized with respect to the mean signal intensity in the un-transfected progenitor cells.

## Measurement of YAP/TAZ staining in vivo

For nuclear quantification, total mean intensity value of the YAP/TAZ staining were measured on the nucleus middle plan with ImageJ software by manual segmentation of the nucleus on the Hoechst channel. The quantification was normalised with respect to the mean signal intensity in the non-transfected progenitor cells or the mean intensity of progenitor cells of the same image. The results are expressed as the ratio of the values of the cells over the mean values of the progenitor cells.

For apical quantification, total mean intensity value of the YAP/TAZ staining were measured on the apical contact plane containing the centrioles with ImageJ software by manual segmentation. The quantification was normalised with respect to the mean signal in- tensity in the non-transfected progenitor cells or the mean intensity of progenitor cells of the same image. The results are expressed as the ratio of the values of the cells over the mean values of the progenitor cells.
Measurement of phospho-RB1 staining in vivo

Total mean of phospho-RB1 intensity of fluorescence were measured with ImageJ software in differentiating ependymal cells at P5 transfected with mb-cherry, on the sum projection of the 5 most central z-stack sections of the nucleus normalized with respect to the mean signal intensity in the three closest un-transfected progenitor cells. The results are expressed as the ratio of the values of the transfected cells over the mean values of the three surrounding control cells.

Statistics

Means of two conditions were compared with the Mann-Whitney test. Means of more than two conditions were analysed by one-way ANOVA followed by Dunn's multiple comparison tests (GraphPad Prism 6.0). Nonparametric test were used, analysis of normality was thus not performed. Analyses of cell distribution at different stages were performed by two-way ANOVA with Sidak's multiple comparison test. Scatter plots are presented as the mean ±sem. ROUT methods were used to remove outliers generated by phal-loidin and YAP/TAZ quantification, with a Q value of 1% (GraphPad Prism 6.0). The number of biological replicates (n), the nature of these replicates and the test performed are indicated in each figure legends.