Materials and Reagents

Microscope slide
Coverslips
Mice at desired stage [between postnatal day 1 (P1) and 21 (P21)]
1x PBS
Triton X-100
4% Paraformaldehyde (PFA) (prepared by dilution of powder in 1x PBS)
Bovine Serum Albumin (BSA) (Sigma-Aldrich, catalog number: A-8022 )
Hoechst 33258 solution (Sigma-Aldrich, catalog number: 94403 )
Mowiol (Merck Millipore Corporation, Calbiochem^®, catalog number: 475904 )
Prolong Gold antifade reagent (Thermo Fisher Scientific, Molecular Probe^TM, catalog number: P36934 )
ZO1 (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 61-7300 )
γ-tubulin [GTU-88] (Abcam, catalog number: ab11316 )
FGFR1OP (FOP) (Abnova Corporation, catalog number: H00011116-M01 )
β-catenin (BD, catalog number: 610153 )
FoxJ1 (eBioscience, catalog number: 14-9965 )
Acetylated-α-tubulin (Sigma-Aldrich, catalog number: T6793 )
Secondary antibodies
1. Anti-Rabbit A647 (Invitrogen, catalog number: A21244 )
2. Anti-Mouse IgG1 A568 (Invitrogen, catalog number: A21124 )
3. Anti-Mouse IgG2b A488 (Invitrogen, catalog number: A21141 )
4. Anti-Mouse IgG1 A488 (Invitrogen, catalog number: A21121 )
5. Anti-Mouse IgG2b A568 (Invitrogen, catalog number: A21144 )
Primary and secondary antibodies (see Recipes)

Equipment

Dissection tools including forceps (Zillow, Dumont, model: 55 ) and Ultra Fine Micro Knives (Fine Science Tools, catalog number: 10316-14 )
Confocal microscope (OLYMPUS, model: Fluoview FV1000 )
STED microscope (Leica Microsystems, model: SP8 3x STED ) equipped with a 100x oil objective (HC-PL-APO; NA 1.40; STED white) and a 592 nm depletion laser

Software

Biotool software (Boutin et al., 2014)

Procedure

The general procedure described here is suitable for staining ependymal cells from lateral wall whole mounts of P1 to P21 mice. However, the last sub-dissection step of the tissue has to be performed before the staining for mice older than P5 and after the staining for mice younger than P5 to ensure an optimal result.

After cervical dislocation of the mice, the brain is removed from the skull.
Dissection of the brain at RT in 1x PBS to reveal the ventricular lateral wall (Figure 2) [a protocol including a video is provided in Mirzadeh et al. (2010)].
Fixation of the whole mount by immersion in 500 μl fresh solution of 4% PFA-0.1% Triton X-100 12 min at room temperature (RT). 
Wash 3 times 10 min in 1x PBS-0.1% Triton X-100 (1 ml) at RT with agitation. 
Sub-dissection of the lateral wall by separating it from the underlying striatum [see Mirzadeh et al. (2010) for a precise description of this step]. Performing this sub-dissection at this step of the protocol improves the staining for tissues older than P5.
Incubation in blocking solution 1x PBS-3% BSA (1 ml) 1 h RT with agitation. 
Incubation with primary antibody diluted in 1x PBS-3% BSA (250-500 μl) over-night RT with agitation. 
Wash 3 times 1x PBS -0.1% Triton X-100 (1 ml) 10 min RT with agitation. 
Incubate with secondary antibody (1:800) and Hoechst (1:1,000) diluted in 1x PBS (For confocal observation of samples) OR with secondary antibody (1:800) diluted in 1x PBS only (for STED observation of samples) (800 μl) 1 h RT with agitation. 
Wash 3 times 1x PBS-0.1% Triton X-100 (1 ml) 10 min RT with agitation. 
For tissues younger than P5: sub-dissection of the LWWM by separating the lateral wall and the underlying striatum in 1x PBS [see Mirzadeh et al. (2010)].
Place the whole mount on the slide with ependymal face up. 
Add 7-8 drops of Mowiol (for confocal observation of samples) or of Prolong mounting medium (for STED observation of samples) directly on the whole mount.
Place a coverslip on the sample.
Keep slides at room temperature, protected from the light, for at least 24 h before imaging.

Representative data

Figure 1. Planar organization of ependymal cells in the lateral wall. A. Schematic representation of the mouse forebrain depicting the localization of the lateral wall of the lateral ventricle. Dashed square highlights the region of interest considered for analysis of ependymal planar polarity. B-C. Schemes represent the planar organization of ependymal cells in the region of interest. B. At the cellular level, ependymal cilia are clustered in a tuft (dotted red line) which localize off center of the apical surface (red arrow). Ciliary beatings (small black arrows) are coordinated. The sum of individual ciliary beatings generates the tuft beating direction (Thick grey arrow). In individual cells, the direction of tuft positioning correlates with the direction of tuft beating (C). At the tissue level, beating direction (grey arrows) and positioning of the ciliary tuft (red arrows) are coordinated between neighboring ependymal cells. D-E. Schematic representation of the lateral (D) and ‘en face’ (E) view of individual ependymal cell organization. D. Multicilia axonemes arise from basal bodies (BBs) anchored perpendicularly to the apical surface of the cells (green). BBs display polarized appendices such as the basal foot (red dot) that indicates the effective beating direction of the cilium (small black arrow). E. Observation of cilia BBs from a top view reveals all ependymal polarity features.

Figure 2. Dissection steps for LWWM preparation. LWWM preparations allows the “en face” observation of ependymal cells. A. Brain after removal from the skull. B. Cut the brain between the two hemispheres (dashed red line) using a microscalpel. C. Remove the cerebellum (dashed red line). D-F. Use a microscapel to reveal the hippocampus (dashed red line). G-H. Insert the microscalpel in the ventricle (arrow) to remove the hippocampus and medial ventricular wall. I. Remove cortical wall following the dashed red line. J. Whole mount ready to be fixed, the lateral wall is outlined by the dashed red line.

Figure 3. Examples of staining on P1, P4, P5 and P21 LWWM Confocal (A-E) and STED (F) images. LWWM stained for A. ZO1 (green) and Acetyl-α- tubulin (red) at P1. B. FoxJ1 (green) and DAPI (blue) at P4. C. β-catenin (green) and Acetyl-α- tubulin (red) at P21. D. β-catenin (green) and ɣ-tubulin (red) at P21. E-F. FGFR1 Oncogene Partner (FOP) at P5. Scale bar: 10 μm in A-B, 15 μm in C-D and 2.5 μm in E-F.

Figure 4. Analysis of planar organization of ependymal cells in the lateral wall (A, B, C). Triple immunostaining for ZO1 (white), FGFR1 Oncogene Partner (FOP, green) and γ-tubulin (red) on P21 LWWM allows the observation of translational and rotational polarities at the cellular and tissular level in a single sample. A. Large field picture displaying the tissue organization. B. Zoom on the cell marked with * in (A). C. Zoom on the BBs patch from cell shown in (B). Analysis of ZO1/FOP/γ-tubulin triple staining using Biotool1 software [described in Boutin et al. (2014)] allows the definition of polarity parameters at BB, cell and tissue level. C’. Definition of individual cilia polarity: each black vector represents the rotational polarity axis of individual BB defined as the direction from FOP (green) to γ-tubulin (red) positive dots. The biotool software calculates the mean BB orientation of the patch to define the cellular rotational polarity axis that corresponds to the beating direction of individual cell (grey arrow). B’-B”. Definition of polarity axis at the cellular level: Outline of cell (green) and BB patch (red). The grey vector in (B’) represents the rotational polarity axis of the cell defined as shown in C’. The red arrow in (B’’) represents the translational polarity axis of the cell defined as direction from the cell center (green cross) to the BB patch center (red cross). A’-A”. Analysis of polarity at the tissue level: The translational and rotational polarity axes are defined for each cell and compared to mean direction of the field (black and red thick arrows). Scale bar: 15 μm in A, 2.5 μm in B and 1 μm in C.

Notes

Freshly made 4% PFA aliquots can be stored at -20 °C up to one year and defrosted just before fixation.
Short fixation time is mandatory for good staining. 12 min fixation is sufficient to fix large and small tissues while preserving the structure and giving the best results. We recommend not to exceed 20 min fixation for ependymal observation.

Recipes

Primary antibodies with the corresponding secondary antibodies

Table 1. List of primary and secondary antibodies

Antibody	Isotype	Reference	Dilution	Secondary antibody
ZO1	Rabbit	Invitrogen 61-7300	1:600	Anti-Rabbit A647
γ-tubulin [GTU-88]	Mouse IgG1	Abcam ab11316	1:400	Anti-Mouse IgG1 A568
FGFR1OP (FOP)	Mouse IgG2b	Abnova H00011116-M01	1:2,000	Anti-Mouse IgG2b A488
β-catenin	Mouse IgG1	BD Transduction Laboratories 610153	1:1,000	Anti-Mouse IgG1 A488
FoxJ1	Mouse IgG1	eBioscience 14-9965	1:2,000	Anti-Mouse IgG1 A488
Acetylated-α- tubulin	Mouse IgG2b	Sigma T6793	1:1,000	Anti-Mouse IgG2b A568

Acknowledgments

The method described in this article has been optimized for detection of ependymal cells organization and is based on a protocol described previously (Mirzadeh et al., 2010). The original version of the protocol has been described in (Boutin et al., 2014). The authors wish to thank the Spassky lab (ENS, Paris) for introduction to the LWWM dissection method. CB is a recipient of a postdoctoral fellowship from ‘La Ligue Nationale Contre le Cancer’. HC acknowledges funding from the Agence Nationale de la Recherche (ANR-13-BSV4-0013-01-ATMIR). This work was supported by the French National Research Agency through the "Investments for the Future" program (France-BioImaging, ANR-10-INSB-04).

References

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