# Dual role of Xenopus Odf2 in multiciliated cell patterning and differentiation

Aude Nommick, Alexandre Chuyen, Raphaël Clément, Virginie Thomé, Fabrice Daian, Olivier Rosnet, Fabrice Richard, Nicolas Brouilly, Etienne Loiseau, Camille Boutin, et al.

► To cite this version:

Aude Nommick, Alexandre Chuyen, Raphaël Clément, Virginie Thomé, Fabrice Daian, et al. Dual role of Xenopus Odf2 in multiciliated cell patterning and differentiation. Developmental Biology, 2025, 520, pp.224-238. 10.1016/j.ydbio.2025.01.014. hal-05355854

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1 Dual role of *Xenopus Odf2* in multiciliated cell patterning and differentiation

2

3 Aude Nommick¹,³, Alexandre Chuyen¹,⁴, Raphael Clément¹, Virginie Thomé¹, Fabrice

4 Daian¹,⁵, Olivier Rosnet¹,⁶, Fabrice Richard¹, Nicolas Brouilly¹, Etienne Loiseau²,

5 Camille Boutin¹, Laurent Kodjabachian¹*

6

7 ¹Aix Marseille Univ, CNRS, IBDM, Turing Centre for Living Systems, Marseille, France

8 ²Aix Marseille Univ, CNRS, CINAM, Turing Centre for Living Systems, Marseille,

9 France

10 ³Present address : Institut Jacques Monod, CNRS - Université Paris Cité, Paris,

11 France

12 ⁴Present address : Department of Chemistry and Biology, Toronto Metropolitan

13 University, Toronto, Ontario, Canada

14 ⁵Present address : Aix Marseille Univ, CNRS, LIS, Marseille France

15 ⁶Present address : Aix Marseille Univ, CNRS, INSERM, Institut Paoli-Calmettes,

16 CRCM, Marseille, France

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18 *Author for correspondence: laurent.kodjabachian@univ-amu.fr

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20 Running title: Odf2 in frog ciliated epidermis

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22 Keywords: Odf2, *Xenopus*, centriole, cilia, polarity, migration

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24 Summary statement: This study shows that the centriolar component Odf2 is

25 required for the correct spacing and differentiation of multiciliated cells in the

26 embryonic epidermis of *Xenopus laevis*.
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Abstract

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In developing tissues, the number, position, and differentiation of cells must be
coordinately controlled to ensure the emergence of physiological function. The
epidermis of the *Xenopus* embryo contains thousands of uniformly distributed
multiciliated cells (MCCs), which grow hundreds of coordinately polarized cilia that beat
vigorously to generate superficial water flow. Using this model, we uncovered a dual
role for the conserved centriolar component Odf2, in MCC apical organization at the
cell level, and in MCC spatial distribution at the tissue level. Like in other species,
*Xenopus* Odf2 localized to the basal foot of basal bodies. Consistently, Odf2
morpholino-mediated knockdown impaired basal foot morphogenesis. Consequently,
the rate of microtubule nucleation by Odf2-deficient basal bodies was reduced, leading
to cilia disorientation, reduced beating, and ultimately altered flow production across
the embryo. Furthermore, we show that Odf2 is required to maintain MCC motility and
homotypic repulsion prior to their emergence into the surface layer. Our data suggest
that Odf2 promotes MCC spacing via its role in the modulation of cytoplasmic
microtubule dynamics. Mathematical simulations confirmed that reduced migration
speed alters the spacing order of MCCs. This study provides a striking example of
coupling between organizational scales by a unique effector.
45 **Introduction**

46 Ciliated epithelia contain multiciliated cells (MCCs), which harbor dozens to hundreds
47 of motile cilia beating coordinately to generate directional fluid flows (Boutin and
48 Kodjabachian, 2019; Mahjoub et al., 2022; Spassky and Meunier, 2017). In many
49 aquatic animals, MCCs ensure locomotion or feeding. In human, MCCs are involved
50 in circulation of the cerebrospinal fluid in the central nervous system, evacuation of
51 soiled mucus from upper airways, and transportation of gametes in genital tracts
52 (Meunier and Azimzadeh, 2016; Spassky and Meunier, 2017).

53 MCC differentiation is a highly regulated multistep process (Al Jord et al., 2014; Al Jord
54 et al., 2017; Revinski et al., 2018; Zhao et al., 2013). First, post-mitotic MCCs must
55 amplify dozens to hundreds of centrioles, using centriole-dependent and deuterosome-
56 dependent (acentriolar) pathways. Next, centrioles disengage from their platform of
57 assembly, move to the apical cell surface, and dock at the membrane to eventually
58 nucleate the formation of motile cilia. The apical migration of centrioles is known to
59 depend on acto-myosin cytoskeleton dynamics (Boisvieux-Ulrich et al., 1990;
60 Lemullois et al., 1988). During this step, centrioles acquire specific appendages, the
61 basal foot (BF) and the ciliary rootlet, after which they are renamed basal bodies (BBs).
62 BF and ciliary rootlets interact with cortical microtubules (MTs) and actin filaments,
63 respectively, thus allowing the formation of an array of uniformly oriented BBs/cilia
64 (Herawati et al., 2016; Werner et al., 2011).

65 While ciliary rootlet biology remains little studied, much more is known about the
66 molecular composition, the architecture and the function of the BF. A pioneer study
67 revealed that the depletion of the centriolar component Odf2 (Outer dense fiber of
68 sperm tails 2)/Cenexin causes the absence of BF in mouse tracheal MCCs (Kunimoto
69 et al., 2012). Consequently, BBs no longer nucleate microtubules and get disoriented,
70 which prevents ciliary beating coordination and alters mucociliary clearance (Herawati
71 et al., 2016; Kunimoto et al., 2012). The role of Odf2 in MCC organization, and
72 polarized beating has been extended to planarians, suggesting strong evolutionary
73 conservation (Basquin et al., 2019). Super-resolution microscopy revealed the modular
74 organization of the BF, with Odf2 being localized near the BB, in a position compatible
75 with a role in scaffolding more distal modules (Nguyen et al., 2020). The mammalian
76 odf2 gene is characterized by extensive alternative splicing, giving rise to multiple
77 isoforms, some of which contain domains required for BF and cilia formation (Kunimoto
78 et al., 2012; Tateishi et al., 2013). Typically, isoforms that promote BF formation are
79 characterized by the presence of a short N-terminal stretch, named Cenexin insertion,
80 and a specific C-terminal extension (Chang et al., 2013; Kunimoto et al., 2012; Tateishi
81 et al., 2013).

82 The mucociliary epithelium that covers the *Xenopus* embryo has provided much of our
83 current knowledge in this field (Boutin and Kodjabachian, 2019; Brooks and
84 Wallingford, 2014; Cibois, 2014; Collins et al., 2021b; Walentek, 2022). The mature
85 epidermis is composed of two cell layers, a basal layer of stem cells and a superficial
86 epithelial layer containing uniformly distributed MCCs and several secretory cell types,
87 including ionocytes that regulate ion and pH homeostasis, goblet cells that produce
88 mucus, and small secretory cells (SSCs) that release antimicrobial peptides and
89 serotonin to modulate ciliary beating frequency. MCCs, ionocytes and SSCs are all
90 born within the mesenchymal layer of the non-neural ectoderm, at the gastrula to early
91 neurula stages, and subsequently intercalate into the surface epithelium. Collectively,
92 MCCs produce flow streamlines that run from dorsal-anterior to ventral-posterior
93 positions, during tailbud and early tadpole life. The alignment of intercalating MCCs
along this axis involves the integration of Planar Cell Polarity (PCP) instructions
mechanically deposited in the epidermis at the end of gastrulation (Chien et al., 2015).
A striking feature of the ciliated *Xenopus* epidermis is the regular distribution of MCCs
over the body. MCCs are born in a disordered pattern, and adopt a regular distribution,
prior to their intercalation into the surface layer (Chuyen et al., 2021b). It was reported
that order emerges from the random walk of MCCs, constrained by mutual repulsion
(Chuyen et al., 2021b). MCCs migrate through the basolateral grooves between
superficial cells, and actively probe intercellular junctions, in preparation for
intercalation at vertices (Chuyen et al., 2021b; Ventura et al., 2022). Consistently,
dynamic remodeling of actin-based lamellipodia was shown to be important for MCC
spacing (Chuyen et al., 2021b), while actin-based filopodia are used to probe vertex
stiffness and select those competent for intercalation (Ventura et al., 2022). Although
MCCs, ionocytes and SSCs all undergo radial intercalation, only MCCs display a
regular pattern of distribution, which may be important for homogeneous flow
production. The physiological function of cilia-powered flows in *Xenopus* has not been
extensively studied. A recent work suggests that water currents flowing over the skin
could limit the risk of exposure to environmental pathogens (Nommick et al., 2022).
Here, we report the first functional study of *Xenopus* Odf2/Cenexin. We confirmed that,
as in mouse and planarian ciliated epithelia, Odf2 localizes to the basal foot and is
required for its assembly and function as a cortical microtubule organizer.
Unexpectedly, we also found that MCC spacing is impaired in absence of Odf2,
presumably because of reduced migration speed and altered homotypic repulsion. Our
data suggest that MCC spacing depends on the function of Odf2 in cytoplasmic
microtubule organization. Thus, the repertoire of Odf2 functions in *Xenopus* has been
expanded to coordinate MCC differentiation and spatial distribution.
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# Results

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## Odf2 is associated to centrioles in *Xenopus* MCCs

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Single cell RNA sequencing in *Xenopus tropicalis* has revealed that *odf2* transcripts are specifically enriched in MCCs of the epidermis, from early neurula stages onwards (Briggs et al., 2018). Consistently, *odf2* expression was also shown to be activated by the MCC regulatory complex Multicilin/E2F/Dp1 in *Xenopus laevis* (Ma et al., 2014).

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Whole mount *in situ* hybridization confirmed that *odf2* transcripts are enriched in MCCs of *Xenopus laevis* epidermis (Fig. S1A,B). In this species, only two Odf2 isoforms are predicted to be encoded from the L genome, both of them containing the N-terminal Cenexin insertion (Fig. S1C). These two isoforms differ in their C-terminus, as isoform 2 contains an additional 178 amino acids. Both isoforms exhibit over 50% identity with human Odf2/Cenexin1 (Fig. S1D). Identity reaches 70% for the C-terminal extension of isoform 2.

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We analysed Odf2 protein subcellular localization at two time points during MCC differentiation, using both a fusion protein between *Xenopus laevis* Odf2 isoform 1 and GFP (Odf2-GFP), and a human Odf2/Cenexin1 antibody raised against a C-terminal polypeptide, highly conserved in isoform 2 (85% identity) (Fig. S1C). In immature MCCs (stage 18), Odf2-GFP displayed a clear association with Centrin-positive centrioles, which at this stage are known to migrate towards the apical cell surface (Fig. 1A). Likewise, endogenous Odf2 was found associated with γ-Tubulin-positive centrioles at stage 18 (Fig. 1C). In mature MCCs (stage 31), Odf2-GFP was tightly associated to BBs docked at the apical surface (Fig. 1B). At this stage, the Odf2-GFP signal appeared nested between Centrin (BB) and γ-Tubulin (BF) signals. The endogenous Odf2 protein displayed the same localization (Fig. 1D). Finally, as already reported in mitotic cells (Hoyer-Fender et al., 2003), both Odf2 and Odf2-GFP were
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detectable at the centrosome, which was most clearly visualized in non-MCCs (Fig. S1E,F).

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Altogether, these data reveal that both *Xenopus Odf2* isoforms are associated to centrioles in epidermal MCCs from the time of their release in the cytoplasm, until maturation into BBs and docking at the apical surface.

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## Odf2 depletion impairs BB docking and orientation

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The localization of Odf2 suggests that it could be involved in centriole/BB organization in MCCs. To investigate this hypothesis, we knocked down Odf2 in MCCs, through injection in the presumptive epidermis of two independent morpholino antisense oligonucleotides (MOs), designed to block translation (*odf2 ATG-MO*), or splicing of the first intron (*odf2 Spl-MO*) (Fig. S2A). The efficiency of *odf2 ATG-MO* was evidenced by the decrease of Odf2 immunofluorescent signals (Fig. S2B). For *odf2 Spl-MO*, its capacity to prevent splicing was confirmed by RT-PCR (Fig. S2C).

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To analyse centriole/BB organization in Odf2-depleted MCCs, we performed immunostaining and transmission electron microscopy (TEM) at stage 31. As previously described, BBs displayed a stereotypical organization in mature control MCCs (Werner et al., 2011). They were all docked, and evenly distributed at the apical surface (Fig. 2A). In contrast, Odf2 knock-down by either MO drastically impaired centriole/BB organization (Fig. 2A). Morphant cells could be classified in two phenotypic categories. In the first category, MCCs were characterized by centrioles/BBs clustered below the cell surface, suggesting deficient apical migration, and/or docking (Fig. 2A). To quantify this defect, we analysed the apical-basal distribution of centrioles along the Z-axis of confocal sections and in transverse sections observed by Transmission Electron Microscopy (TEM). Immunofluorescent

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169 staining showed that in control cells, centrioles localized on average 2µm below the
170 first Centrin-positive confocal slice. In contrast, in *odf2* morphant cells centrioles
171 localized deeper in the cytoplasm, with *odf2* Spl-MO causing more severe defects (Fig.
172 2B). Importantly, docking at the apical surface was efficiently rescued by co-injection
173 of a synthetic form of *odf2* mRNA, that cannot be bound by either *odf2* MOs (Fig. 2B
174 and Fig. S2D,E). TEM images also revealed BBs below the membrane in *odf2* Spl-MO
175 MCCs, which often lied horizontally, in a position incompatible with apical docking (Fig.
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177 In the second phenotypic category, centrioles/BBs were properly localized at the apical
178 surface (Fig. 2C). In such cases, BB orientation could be revealed by double
179 immunostaining against Centrin and γ-Tubulin, which allowed us to automatically
180 extract orientation vectors and plot their circular distribution (Fig. 2C). In control cells,
181 vector angles tightly distributed around the mean, indicating that BBs oriented in the
182 same global direction. In contrast, in *odf2* morphant MCCs, vector angles were widely
183 distributed around the mean, indicating a randomization of BB orientation (Fig. 2C).
184 Accordingly, circular standard deviation (CSD) values were significantly higher in
185 morphants, as compared to control (Fig. 2D). The Rayleigh statistical test also revealed
186 a higher percentage of morphant MCCs, in which no significant mean vector could be
187 defined, as compared to control (Fig. 2E). Again, correct BB orientation was efficiently
188 rescued in both types of morphant MCCs by co-injection with *odf2* mRNA (Fig. 2D,E
189 and Fig. S2D,E). To quantify orientation on TEM images, we drew a vector from the
190 centre of the BB to the tip of the BF and analysed the angular mean deviation. In control
191 situation, these angles tightly distributed around the mean. In morphant cells,
192 randomization of BB vector directions resulted in a wide distribution of angles around
193 the mean (Fig. 3B,D).
As both *odf2* ATG and Spl morphant MCCs could be rescued by *odf2* mRNA injection, establishing specificity, subsequent experiments were performed with one or the other MO, as stated.

Immunostaining against acetylated-Tubulin revealed that ciliogenesis was maintained when BB docking occurred correctly in *odf2* Spl morphant MCCs (Fig. S3A). Altogether, these data show that Odf2 is required for correct apical migration/docking of BBs, as well as for their correct orientation at the apical surface.

## Odf2 depletion impairs basal foot formation

The BF and ciliary rootlet are key appendages required for BB organization at the apical surface of MCCs (Basquin et al., 2019; Herawati et al., 2016; Kunimoto et al., 2012; Wright et al., 1983). Previous studies have established the importance of Odf2 in BF formation, and microtubule nucleation in murine MCCs (Kunimoto et al., 2012). The localization of Odf2 and the phenotypes observed in morphant cells suggest that this protein could also participate to BF formation in *Xenopus* MCCs. To evaluate this possibility, we have scored the presence and measured the dimensions of BF on apical TEM sections. We detected a decrease of 10% points in the rate of presence of BF in *odf2* Spl morphant MCCs, compared to control (Fig. 3E). Furthermore, dual axis serial electron tomography followed by 3D reconstruction and segmentation revealed that BF in *odf2* morphant MCCs (n=22) displayed significantly reduced dimensions, compared to control (n=8) (Fig. 3F,G). This method also revealed that fewer microtubules (MTs) grew from *odf2* morphant BF, compared to control (Fig. 3F,H; Movie 1,2).

Finally, we asked if the other BB appendage, the rootlet, was impacted by Odf2 knockdown. Immunostaining against *Xenopus* Pericentrin (Pcnt), which has been
validated as a bona fide rootlet marker in a previous study (Nommick et al., 2022),
revealed no difference in intensity or localization between control and odf2 ATG
morphant cells (Fig. S3B,C). This suggests that Odf2 is not required for rootlet
formation in Xenopus MCCs.

To conclude, Odf2 is a BB component necessary for the correct assembly of the BF in
*Xenopus*, like in mouse or planarians.

## Odf2 depletion disturbs apical cytoskeleton organization

Next, we evaluated whether defective BB organization in *odf2* morphant cells was
associated to impaired apical cytoskeleton network formation.

Based on the TEM analysis described above, we first analysed the apical MT network,
which was shown to be necessary for proper BB orientation (Werner et al., 2011). In
mature MCCs, α-Tubulin immunostaining reveals cilia, thus precluding visualization of
intracellular MTs. To circumvent this limitation, we adopted a deciliation strategy to
deplete cilia-associated signals, and visualize only intracellular MTs (Nommick et al.,
2022). At stage 31, control cells were characterized by a network of apical MTs
connecting BBs together (Fig. 4A,A'). It can be noted that most BBs appear to organize
several filaments in different directions, similar to what EM tomography revealed (Fig.
3F). Focusing on regions of *odf2* Spl morphant cells, in which apical migration of BBs
was correct, we noticed a strong reduction of MT signals (Fig. 4B,B'). This correlated
with the reduced amount of MTs emanating from *odf2* morphant BBs, as revealed by
EM tomography (Fig. 3F,H).

We next analysed F-actin network in MCCs at stage 31. In control cells, we observed
the stereotypical organization in apical and subapical networks that was previously
described for mature MCCs (Werner et al., 2011). The apical actin network was
organized like a grid surrounding each centriole, and the subapical network located just below was composed of short actin fibres that are known to connect neighbouring BBs (Fig. S4A,A'). Focusing on regions of *odf2* ATG morphant cells, in which apical migration of BBs was correct, we noticed a strong reduction of apical and subapical actin signals (Fig. S4B,B'). We conclude that Odf2, either directly or indirectly, is involved in the polymerization of the apical actin network necessary for the correct spacing of BBs.

Finally, we analysed the apical intermediate filament network using anti-cytokeratin C-11 antibody (Nommick et al., 2022). At stage 31, intermediate filaments were organized into a dense grid surrounding each BB (Fig. S4C,C'). Although intermediate filaments were still visible, the lattice morphology and organization appeared altered in *odf2* Spl morphant cells, which could suggest a defect secondary to BB disorganization (Fig. S4D,D').

Our observations indicate that in *Xenopus* MCCs, like in mouse and planarian MCCs, Odf2 is necessary for the proper organization of apical MTs, through their association to the BF of BBs. Furthermore, this analysis reveals that Odf2 depletion impacts the global organization of MCC apical cytoskeletal networks.

## Odf2 depletion reduces ciliary beating and impairs fluid flow

The disorganization of BBs in *odf2* morphant MCCs is expected to reduce the hydrodynamic forces generated by cilia, and consequently to alter the production of fluid flows at the surface of the embryo.

To address this issue, we first analysed ciliary beating frequency (CBF) by high-speed video-recording. As previously described, control MCCs performed clearly synchronised effective and recovery strokes (Werner et al., 2011). In *odf2* Spl
269 morphant MCCs, cilia beating was often uncoordinated causing collisions, while some 270 cilia were immobile (Movie 3). Consequently, the CBF, averaged per individual MCC, 271 was about 5 Hz lower in morphant embryos, compared to controls (Fig. 5A). We did 272 not, however, observe morphant MCCs where all cilia were immobile, suggesting that 273 Odf2 is not strictly required for beating. Secondly, we analysed cilia-powered fluid flow 274 by live-recording of visible dyed microsphere movement along the flanks of tadpoles 275 (Kulkarni et al., 2018; Nommick et al., 2022). In control condition, microspheres were 276 moved by a robust flow and travelled the entire length of the embryo in approximately 277 12s (Fig. 5B,C, and Movie 4). As expected, an impaired flow was observed in *odf2* 278 morphant condition. In most cases, microspheres only travelled about half the length 279 of the embryo and eventually dispersed in the surrounding medium (Fig. 5B,C and 280 Movie 4).

281

282 Odf2 depletion impairs MCC spacing pattern

283 So far, we have documented the importance of Odf2 in MCC differentiation and activity.
284 Unexpectedly, we also uncovered a role for Odf2 in regular MCC spacing. Adjacent
285 MCCs were observed, at much higher frequency in *odf2* Spl morphant, than in control
286 embryos (Fig. 6A-C). This effect was most prominent at stage 20 (Fig. 6A,C), but
287 remained visible at stage 31 (Fig. 6B,C). In contrast, the rate of radial intercalation of
288 MCCs, which was partial at stage 20, fully recovered by stage 31, upon Odf2
289 knockdown (Fig. 6D). Taken together, these observations indicate that the spacing
290 defects observed in morphants are unlikely to reflect a simple developmental delay.
291 Interestingly, fluorescent tracing revealed that aberrant MCC spacing was more
292 frequent in regions where the *odf2* MO reached the internal layer, suggesting a cell-
293 autonomous effect (Fig. S5A). Importantly, MCC spacing in morphant embryos was
294 rescued by co-injection of *odf2-GFP* mRNA (Fig. 6F). To better quantify MCC spacing, 295 we used Delaunay triangulation to calculate the MCC order index (Chuyen et al., 296 2021b). The order index was significantly decreased in *odf2* morphant embryos, 297 compared to control, indicating that MCCs no longer dispersed at regular intervals in 298 absence of Odf2 (Fig. 6E).

299 These data reveal that Odf2 is required in MCCs for a correct spacing pattern to be 300 established.

301

302 Odf2 is necessary to maintain optimal MCC migration

303 We previously reported that MCCs disperse at regular intervals through random 304 migration and homotypic repulsion, within the inner layer of the ectoderm, prior to radial 305 intercalation (Chuyen et al., 2021b).

306 Based on its localisation at the centrosome (Figure S1E,F) and its ability to interact 307 with microtubules (Donkor et al., 2004), we hypothesised that Odf2 could promote the 308 dynamics of the cytoplasmic microtubule network, thereby assisting MCC movement 309 and spacing. Supporting this hypothesis, we found that the formation of cytoplasmic 310 microtubules, revealed by α-Tubulin immunofluorescent staining, was severely 311 impaired in *odf2* morphant MCCs, prior to radial intercalation (Fig. S6A,B). 312 Furthermore, microtubule depolymerization, caused by Nocodazole treatment during 313 the phase of MCC migration, resulted in irregular MCC distribution, despite their correct 314 radial intercalation (Fig. S6C-E).

315 To directly visualize and quantify MCC migration during the planar walking phase, we 316 used time-lapse confocal video-microscopy on stage 14 embryos expressing LifeAct- 317 GFP, under the control of the MCC-specific promoter *α-tubulin* (Chuyen et al., 2021a; 318 Chuyen et al., 2021b). Control MCCs displayed a typical behaviour, characterized by
random movement, multidirectional extension of cellular protrusions, and mutual repulsion following contact (Fig. 7A, and Movie 5). In contrast, *odf2* Spl morphant MCCs appeared to migrate less actively, and to maintain prolonged contacts (Fig. 7A, and Movie 5). The quantification of instant speed recorded every minute over a period of 4 hours, revealed that *odf2* morphant MCCs travelled at an average speed of 0,26 µm/min, only about 40% the average speed of control MCCs (0,66 µm/min) (Fig. 7B). Consequently, the average cumulative distance travelled by *odf2* morphant MCCs (50 µm), and their average displacement between the start and the end of the recording (20 µm) was dramatically reduced, when compared to control cells (160 µm average cumulative distance, and 50 µm average displacement) (Fig. 7C,D). Moreover, the average duration of mutual contacts between MCCs was increased, by about 5-fold in *odf2* morphant (150 min) vs control condition (30 min) (Fig. 7E). Consequently, 40% of the morphant MCCs remained in contact during the 4 hours of recording, whereas it never happened in the control condition. mRFP injection revealed that control, as well as *odf2* morphant MCCs, maintained a tight association to outer-layer cell junctions during migration (Fig. 7F) (Chuyen et al., 2021b).

Our analyses suggest that reduced migration speed, and extended contact times could explain the irregular MCC spacing visible in *odf2* morphant embryos, following the completion of radial intercalation.

In a previous study, we designed an energy-based mathematical model, which confirmed that reduced mutual repulsion between MCCs caused a significant degradation of the order index (Chuyen et al., 2021b). Thus, we used this model to specifically evaluate the consequence of reducing MCC migration speed. To do so, the capacity for mutual repulsion and affinity for outer-layer cell junctions were maintained at the same levels as the control situation, and MCC migration speed was reduced 2-
344 fold, similar to our experimental measurements (Fig. 7B). Strikingly, reducing migration
345 speed caused a significant decrease of the MCC order index (Fig. 7G and Fig. S7;
346 Movie 6), comparable to the decrease measured between control and *odf2* morphant
347 conditions (Fig. 6E).

348 Based on our experiments and simulations, we conclude that Odf2 **may promote**
349 **microtubule dynamics** to maintain an optimal migration speed of MCCs, such that they
350 can move away from each other and regularly disperse, by the time they need to
351 radially intercalate.

352
353 **Discussion**

354 In this paper, we report the first functional study of the centriole component
355 Odf2/Cenexin in *Xenopus*. Like in mouse and planarian MCCs (Basquin et al., 2019;
356 Kunimoto et al., 2012), *Xenopus* Odf2 localizes at the BF of BBs, and is necessary for
357 BF assembly, MT nucleation, BB orientation, polarized cilia beating and fluid flow
358 production across the embryo. Strikingly, Odf2 is also required for optimal migration
359 speed and spacing of MCCs, prior to their intercalation into the surface cell layer of the
360 epidermis.

361 Two distinct Odf2 isoforms are predicted from the genome of *Xenopus laevis*, which
362 differ in their C-terminus, as isoform 2 contains an additional stretch of 178 amino-
363 acids. The position and sequence of this extension correspond to the C-terminal
364 extension present in mammalian Odf2/Cenexin isoforms, which were shown to be
365 involved in centriole/BB architecture and function (Chang et al., 2013; Herawati et al.,
366 2016; Ishikawa et al., 2005; Kunimoto et al., 2012; Tateishi et al., 2013).
367 Immunostaining with an antibody raised against this extension confirmed that isoform
368 2 localizes at centrosomal centrioles, as well as at BBs in MCCs. Interestingly, isoform
369 1 displayed the same localization, when fused to GFP. Like isoform 2, isoform 1
370 contains the N-terminal Cenexin insertion, which was shown to be necessary for
371 centrosomal localization (Huber et al., 2008). Our data thus suggest that isoforms 1
372 and 2 colocalize in centrosomes and BBs, and may act in concert in somatic cells,
373 particularly in MCCs. Transcripts for both isoforms are also expressed in adult
374 *Xenopus* testis (Fisher et al., 2023), consistent with the role of Odf2 in the maintenance
375 of sperm tail integrity (Ito et al., 2019; Tarnasky et al., 2010).

376 Pioneer studies in mouse and planarian MCCs have established a crucial role for Odf2
377 in the construction of the basal foot (Basquin et al., 2019; Kunimoto et al., 2012), an
asymmetric appendage essential to nucleate MTs, which in turn help to link and
uniformly orient BBs (Herawati et al., 2016; Werner et al., 2011). Our findings in
*Xenopus* largely converge with those studies, further confirming that the biological
function of Odf2 in MCCs is conserved across animal evolution. Immunofluorescent
staining shows that the Odf2 signal is nested between Centrin (BB) and γ-Tub (distal
tip of the BF) signals, supporting its localization at the base of the BF. Consistent with
this proximal position, removal of Odf2 reduces BF dimensions, in agreement with its
proposed scaffolding function (Basquin et al., 2019; Kunimoto et al., 2012; Nguyen et
al., 2020). Consequently, the rate of MT nucleation is decreased in *odf2* knocked down
MCCs, and BB rotational polarity is randomized. Interestingly, Odf2 depletion in
planarian MCCs does not randomize BB orientation, but instead uniformly modifies
their angle of rotation (Basquin et al., 2019). Similarly, despite the complete absence
of BF in Odf2 mouse mutant tracheal MCCs, the orientation of cilia does not appear
totally randomized (Kunimoto et al., 2012). This difference may reflect stronger
physical links between BBs in mouse and planarians MCCs, as suggested by their
alignment into rows that are not readily apparent in *Xenopus* epidermal MCCs.

Similar to mouse and planarian, rootlet formation and ciliogenesis is unaltered in
absence of Odf2 in *Xenopus* MCCs. Although ciliary beating is maintained, it appears
erratic and, on average, slower than in control MCCs. This is likely due to cilia
disorientation preventing the correct deployment of effective and recovery strokes. The
alteration of ciliary beating upon Odf2 depletion appears stronger in *Xenopus* than in
mouse and planarian MCCs (Basquin et al., 2019; Kunimoto et al., 2012), perhaps
again reflecting more dramatic cilia disorientation. Altered ciliary beating ultimately
impairs fluid flow along the embryo. Although not tested here, this is expected to
reduce pathogen clearance and survival (Nommick et al., 2022).
We further report that Odf2 depletion not only alters the MCC apical MT network, but also dramatically reduced the apical and sub-apical actin networks. This suggests that the BF is important to stabilize actin filaments in the BB lattice. Supporting this view, sub-apical actin fibres appear to link the distal tip of the striated rootlet of each BB to the immediately anterior BB, at a position compatible with the BF (Werner et al., 2011). Another supporting evidence comes from a study of Zeta-Tubulin, which localizes at the distal tip of the BF, and is necessary to stabilize sub-apical actin filaments but not MTs (Turk et al., 2015). Finally, this view is supported in a study of mouse ependymal MCCs, which reported that the BF component Anks1a is necessary to stabilize apical MTs, as well as apical and sub-apical actin filaments (Ryu et al., 2021). Thus, the BF appears to play a critical role in organizing the apical MCC cytoskeleton and the BB network.

We detected Odf2 in association to newly synthesized centrioles at a stage when they are known to migrate towards the apex of the MCC. Consistently, in the most severely affected *odf2* morphant MCCs, many centrioles fail to reach the apical surface, a phenotype also reported for mouse tracheal MCCs lacking Odf2 (Kunimoto et al., 2012). Odf2 has been shown to be a Microtubule-Associated-Protein (MAP), which helps the anchorage of MTs to centriole appendages and modulates MT dynamics (Donkor et al., 2004; Hung et al., 2016). As the process of centriole apical migration is known to chiefly depend on acto-myosin cytoskeleton dynamics (Boisvieux-Ulrich et al., 1989a, 1990; Ioannou et al., 2013; Lemullois et al., 1988), it is not immediately obvious how Odf2 could contribute to this process as a MAP. A provocative hypothesis would be that dynamic MTs nucleated by newly formed centrioles loaded with Odf2, would assist apical migration. This view is consistent with the observation that MT stabilization by Taxol impairs apical migration of centrioles in quail oviduct MCCs
(Boisvieux-Ulrich et al., 1989b). Alternatively, it is formally possible that BF-associated
proteins, such as Zeta-Tubulin, are already present in neo-synthesized centrioles, to
help actin-dependent apical migration. ScRNAseq data are compatible with this
hypothesis as both *zeta-* and *epsilon-tubulin* transcripts are enriched in immature
*Xenopus tropicalis* epidermal MCCs, at the time of centriole apical migration (Briggs et
al., 2018; Turk et al., 2015).

In *Xenopus* embryonic epidermis, centriole apical migration occurs in immature MCCs,
while they transit from the inner layer to the surface layer, where they can terminate
differentiation and engage ciliary beating (Collins et al., 2021b). A significant body of
work established an important role for MTs and MAPs in radial intercalation (Collins et
al., 2021a; Collins et al., 2020; Werner et al., 2014). Specifically, it was shown that the
timing of MCC and ionocyte insertion can be accelerated or delayed by increasing or
decreasing the number of MTs, or their level of acetylation (Collins et al., 2021a; Collins
et al., 2020). Strikingly, as centrioles can nucleate MT growth, their number also
modulates the timing of radial intercalation, explaining why MCCs normally intercalate
before ionocytes (Collins et al., 2020). We observed that the formation of cytoplasmic
microtubules was impaired in *odf2* morphant MCCs, providing a potential explanation
for their delayed radial intercalation.

After their apical migration, BBs must dock at the cell membrane, a process that also
appears to be incomplete in *odf2* morphant MCCs (Figure 3A). Here, it is interesting to
note that Odf2 was shown to bind the protein Chibby, at the distal appendages of the
mother centrosomal centriole (Steere et al., 2012). Chibby was later shown to be
required for apical BB docking in tracheal MCCs, through its role in the recruitment of
the ciliary vesicle necessary for the fusion with the plasma membrane (Burke et al.,
2014). *Xenopus tropicalis chibby* transcripts are enriched in immature MCCs (Briggs et al., 2018), consistent with a possible role of Odf2 and Chibby in apical docking.

Our study reveals that Odf2 is required for regular spacing of MCCs, a process known to depend on the random walk of immature MCCs, constrained by homotypic repulsion, prior to intercalation (Chuyen et al., 2021b). This is to our knowledge the first report of a functional link between Odf2 and cell migration. Our evidence clearly supports an MCC-autonomous involvement of Odf2 in the spacing process. Using time-lapse confocal video-microscopy, we could quantify the instant speed, time of contact, and distance travelled by a large number of control and *odf2* morphant MCCs. This analysis reveals that Odf2-deficient MCCs move at about only half the speed of control MCCs, and remain in contact after homotypic collisions about five times longer. Consequently, Odf2-deficient MCCs travel much shorter distances than their control counterparts and fail to disperse. Similar to control, Odf2-deficient MCCs move through the grooves between outer layer cells, suggesting that MCC affinity for outer-cell junctions, mediated by the Scf/Kit pathway (Chuyen et al., 2021b), is not altered by Odf2 depletion. Our mathematical model confirms that reducing MCC speed is sufficient to degrade their order index, even when homotypic repulsion is maintained, to an extent comparable to the order index measured in *odf2* morphant embryos. This suggests that reduction in MCC migration speed caused by Odf2 depletion is the major factor contributing to pattern degradation. Our previous work showed that migration speed is highest during the first 3-4 hours of the planar walking phase, before MCCs slow down, and stabilize under vertices in preparation for intercalation (Chuyen et al., 2021b). Accordingly, this faster walking phase contributes most significantly to improve the MCC order index (Chuyen et al., 2021b). Odf2-deficient MCCs move at a speed normally observed during the slower phase of the process, which contributes much
less to improve the MCC order index (Chuyen et al., 2021b). Based on this data, we formulate a model whereby MCC migration speed is optimized to allow spacing in the short time-window available before they switch to vertically-directed movement, in preparation for intercalation. As discussed above, Odf2 knockdown delays MCC intercalation, possibly giving extra time to improve order. However, in *odf2* morphant embryos, the MCC spacing pattern and the order index are still much lower than control, after completion of intercalation. This suggests that the migration speed of MCCs lacking Odf2 is too low to efficiently convert the extra time available into a significantly improved spacing pattern. Interestingly, our mathematical model also suggests that the order index plateaus along the fictive time of the stepwise simulation, and that slower MCCs can never catch up their control counterparts.

Based on our results and on the knowledge accumulated in previous studies, we propose that Odf2 contributes to optimize MCC spacing through its capacity to modulate MT dynamics. A large body of work has established the importance of the crosstalk between MTs and other cytoskeletal elements in cell migration (Bouchet and Akhmanova, 2017; Seetharaman and Etienne-Manneville, 2020). In particular, it has become clear that MTs are necessary to promote actin-dependent lamellipodia protrusions that are essential for migration of mesenchymal cells. During the walking phase within the inner epidermal layer, MCCs do use a mode of migration based on actin-dependent lamellipodia (Chuyen et al., 2021b). It is interesting to note that when lamellipodia formation is prevented through exposure to the Arp2-3 inhibitor CK-666, MCC migration speed is twice lower than control, time of mutual contact is 4 times longer, but outer-cell junction-guided migration is preserved (Chuyen et al., 2021b). In other words, CK-666 and *odf2* knockdown have identical consequences, suggesting that Odf2 may be required to maintain actin-dependent lamellipodia-based MCC
migration and homotypic repulsion. This function in MCC movement is likely to mobilize
the pool of centrosomal Odf2, as it is known to modulate MT organization and stability
(Hung et al., 2016). In contrast, it is unlikely that Odf2 at neo-synthesized centrioles
participates to this process, as MCCs most actively move several hours before
centriole amplification is engaged (Revinski et al., 2018).

In conclusion, this work reveals a dual role for Odf2 in organizing the MCC ciliary
carpet, and in promoting regular MCC distribution, in the *Xenopus* mucociliary
epidermis. It provides a striking example of functional integration at different
organizational scales of a unique conserved effector, which allows adaptation to the
specific constraints of a system. It is currently impossible to determine whether fluid
flow production is dependent on the two functions of Odf2 uncovered in this study.

Future work should therefore address with more specific tools if regular MCC
distribution contributes to fluid flow production and pathogen clearance.
## 515 Materials and methods

### 517 Ethics statement

All experiments were performed following the Directive 2010/63/EU of the European parliament and of the council of 22 September 2010 on the protection of animals used for scientific purposes and approved by the "Direction Départementale de la Protection des Populations, Pôle Alimentation, Santé Animale, Environnement", des Bouches du Rhône (agreement number F1305521).

### 524 Xenopus embryo culture, injection and fixation

Ovulation was stimulated in *Xenopus laevis* adult females from NASCO (https://www.enasco.com) by injection of (800 units/animal) Human Chorionic Gonadotropin (ChorulonR). On the following day, eggs were recovered by gentle massaging, fertilized *in vitro* with sperm from NASCO males, de-jellied in 2% cysteine hydrochloride (pH 8.0) and washed, first in water, then in 0.1X MBS (Modified Barth's Saline). At 2-cell stage, embryos were transferred in 4% Ficoll-MBS 1X. Injections were performed using needles made from pulled glass capillaries and mounted on a Nanoject II injector (Drummond).

mRNA expression constructs were injected at different concentrations and stages (Table 1). For MOs, 20 to 30 ng was injected (alone or with 200-500pg of mRFP or GFP-gpi tracers) in one or two animal-ventral blastomere (presumptive epidermis) at 4- or 8-cell stage. Non-toxic MO concentrations were determined by analyzing embryo survival rates. For the rescue of BB docking and orientation, a sequential injection strategy was adopted to obtain mosaic embryos containing morphant and rescued MCCs: (1) at 4-cell stage, *odf2* ATG or Spl MO + Centrin-RFP was injected, then (2)
at 16-cell stage, the same embryos were injected with *odf2-GFP* mRNA (construct resistant to both *odf2* MOs). For the rescue of MCC spacing, *odf2* MOs and *odf2-GFP* mRNA were co-injected at 4-cell stage to limit mosaicism.

After injection, embryos were incubated at 13°, 18° or 23°C in MBS 0.1x until they reach the desired developmental stage. Whole embryos were fixed with different fixatives (Table 2) and conserved in 100% methanol at -20°C.

## Whole-mount chromogenic In Situ Hybridization (WISH) and whole-mount Fluorescent In Situ Hybridization (FISH)

To synthesize the *odf2* riboprobe, a cDNA fragment from *Xenopus laevis* *odf2.L* was amplified from a commercial clone (gene ID 496006, Horizon Discovery) by PCR using the following primers:

*odf2* forward: 5'-GCGGCCGCTTGGACATCACACCTCCA -3'

*odf2* reverse: 5'-GAATTCACGAGCTGCCTGTTAAGCAT -3'

The *odf2* PCR product was cloned in pBlueScript KS+ vector, and mRNAs were synthesized *in vitro* and labelled with digoxigenin-UTP, using the mMessage mMachine T7 or T3 transcription kits (Ambion). Finally, RNA antisense and sense (as negative control) probes were purified with Macherey-Nagel NucleoSpin RNA Clean-up kit and used at 1ng/µl.

WISH was performed as previously described (Marchal et al., 2009), with 40 ng of *odf2*-DIG riboprobe. Pictures were taken with a stereomicroscope Leica MZ125 coupled to NIKON digital Sight DS-Fi1 camera. *Alpha-tubulin* FISH was performed as previously described (Castillo-Briceno and Kodjabachian, 2014).
564 Expression constructs and morpholinos

565 The complete *Xenopus laevis* *odf2* ORF was amplified by PCR from a commercial
566 clone (gene ID 496006, Horizon Discovery) using the following primers:

567 *odf2*-forward: 5' TCTTTTTGCAGGATCACAATGAAAAACCGCTCTCCCTCTC 3';

568 *odf2*-reverse: 5' CTTTACTCATTCTAGACAGGGCACAGTTGCGCCAAT 3';

569 The PCR product was cloned in frame with GFP in 3' into pCS2+-GFP vector using In-
570 Fusion® HD Cloning Kit (Takara Bio USA, Inc.). Capped mRNA was synthetized from
571 the linearized construct using the SP6 mMessage mMachine Kit (Ambion) and purified
572 with the MEGAclear™ Kit (Ambion).

573 Two independent morpholino antisense oligonucleotides were designed against *odf2*
574 (GeneTools, LLC) (Fig. S2A):

575 *odf2* ATG-MO: 5' GAGAGCGGTTTTTCATTGCCCTCTT 3'

576 *odf2* Spl-MO: 5' AGTATCTGGTTATTTCCCACCTGTG 3'.

578 PCR for *odf2* Spl-MO Validation

579 The efficiency of the *odf2* Spl-MO in blocking splicing has been confirmed by RT-PCR.

580 First, total RNA was extracted from non-injected and *odf2* Spl-MO-injected embryos
581 (n=10 embryos/condition) at stages 10/14/20 using RNeasy mini Kit Qiagen. Then,
582 cDNA was produced with the iScript Reverse transcription Supermix (Biorad). *odf2*
583 Spl-MO targets the Exon1-Intron1 junction. To validate its efficiency we have amplified
584 the cDNA (with Platinum HiFi DNA Polymerase) with primers targeting exon 1 and
585 exon 2 (EurofinGenomics) (Fig. S2A):

586 Forward: 5'- GGGCAATGAAAAACCGCTCT - 3' (exon 1)

587 Reverse: 5'- CACAGTTGCACTCCTCCTGA - 3' (exon 2)

588 As control, genomic DNA from *Xenopus laevis* embryo and A6 cells was also amplified.
589

590 Immunostaining

591 Embryos were fixed at the desired stage in different fixatives depending on the primary
592 Ab used (Table 2). After three washes in PBS 1X embryos were incubated in blocking
593 solution (3% Bovine Serum Albumin in PBS 1X) for 1h at RT and subsequently in
594 primary Abs diluted in blocking solution O/N at 4°C (Table 2). After washing in PBS 1X
595 embryos were incubated with fluorescently-labelled secondary Abs (Table 3) diluted in
596 blocking solution for 1h at RT. After washing, embryos were mounted in Mowiol
597 (Sigma-Aldrich) between slide and coverslip.

598

599 Fluorescent image acquisition

600 Images of immunostained and FISH-stained embryos were acquired using ZEISS LSM
601 780 inverted Axiolmager Z2 and ZEISS LSM 880 reverse standing AxioObserver 7
602 equipped with 20X and 63X Oil objectives. Two or three-colors confocal z-series
603 images (z-slices interval between 0.3-0.8nm depending on the experiment) were
604 acquired using sequential laser excitation. When necessary, images were converted
605 into single plane maximum intensity projection (MIP) and processed using Fiji or
606 ImageJ softwares.

607 To calculate the MCC order index, the outlines of individual *alpha-tubulin*-positive cells
608 were manually drawn from MIP using the FIJI 'ROI manager' tool. This allowed to
609 position MCC centroids and to calculate the order index, based on Delaunay
610 triangulation, as described in the section 'Mathematical Modelling'.

611 To quantify α-Tubulin signal intensity in MCCs, ROI were manually delineated on
612 confocal stacks around basal body clusters and quantified using ImageJ. Background
613 signal was obtained by drawing ROI in random areas deprived of MCCs. Finally, the
614

α-Tubulin net mean signal intensity (MSI) values in MCCs were obtained by subtracting
the mean of the background from the values measured in MCCs.

615

616

Live microscopy was performed as previously published (Chuyen et al., 2021a). Time-lapse imaging was done at 23°C starting at stage 14-15 and for a duration of 2 to 4 h on a Nikon Roper Spinning-disk Eclipse Ti inverted microscope using a 20X objective.

617

618

619

Image acquisition was performed with the Meta-Morph software. 20µm-deep Z
sections were acquired every min in 0.5 to 0.7µm steps, averaging 2. To compensate
for fluorescence intensity variations across different injected embryos, the laser power
was adjusted for each experiment. Excitation wavelengths were 488nm and 561nm for
GFP- and RFP-tagged protein, respectively.

620

621

622

623

624

625

Transmission electron microscopy (TEM)

626

627

628

629

630

631

632

633

Embryos at stage 31 were fixed with glutaraldehyde 2.5 %, PAF 2 % in Cacodylate buffer 0.1 M pH 7.4, and post-fixed with 1 % Osmium Tetraoxyde in Cacodylate buffer 0.1 M pH 7.4 during 1 hour at RT. They were dehydrated in alcohol and embedded in Epon. Next, embryos were cut transversely at the mid-body zone (80 nm thick sections) and longitudinally at embryo flanks (80 nm thick sections, or 350 nm thick sections for tomograms) with a Leica Ultracut UC7 (Leica, Germany). Images were acquired using a Tecnai G2 (Thermofisher, USA) microscope equipped with a Veleta camera (Olympus, Japan).

634

635

636

637

638

For electron tomography, tilt series were taken at different angles following the saxton
scheme. The ETomo software (University of Colorado, Boulder) was used for the
treatment of the dual axis tilt series, the tomograms reconstructions and the stitching
of consecutive tomograms. MTs were manually segmented in IMOD and the model
was exported to Amira for visualization. BBs were segmented in Fiji using the Weka
639 3D segmentation plug-in and the probability maps were exported to Amira for
640 visualization and movie realization.

641

642 BB docking/orientation and BF presence quantifications

643 Pcnt signals were quantified by manually tracing the cell contour and measuring the
644 mean pixel intensity per MCC with ImageJ.

645 BB docking was quantified by two methodologies. On immunostaining acquisitions, it
646 was quantified manually by counting the number of z-slices containing Centrin Ab or
647 Centrin-RFP positive centrioles along the apical-basal axis for each cell. These values
648 where transformed in µm based on the interval size between slices set during the
649 confocal acquisition. Secondly, the number of BBs not anchored at the apical surface
650 were manually quantified on transversal TEM sections.

651 BB orientation was analyzed on confocal immunostaining images, as previously
652 reported (Nommick et al., 2022). Circular standard deviation was computed (CSD,
653 Rayleigh's Uniformity Test) with the Oriana software. As BF absence is one of the
654 phenotype caused by Odf2 depletion, only cells presenting a milder phenotype with a
655 clear signal of γ-Tubulin at the BF have been included in the BB orientation
656 quantification.

657 BB orientation was quantified on TEM sections with the Biotool1 software. First, a
658 vector was manually traced from the BB center to the BF tip. Then, the overall average
659 angle between all BBs was calculated automatically and the deviation from the average
660 for each BB was plotted.

661 BF presence and ultrastructural features were quantified on TEM images manually,
662 based on the presence or absence of the typical BF triangular shape protruding from
663 centrioles. Considering that each TEM section has a thickness of 80 nm and that the
total height of a centriole is ~ 500nm, there is ~ 60 % probability of cutting below or over the BF. This baseline value is expected to be the same in control and morphant cases. BB ultrastructural features, such as BF length (vector from centriole center to BF tip), thickness (number of z-slices containing BF material transformed in µm based on the interval size between slices) and width (vector between the two sides of the BF proximal base) was analysed manually on tomograms with ImageJ.

## Chemical treatments

To achieve deciliation and optimize the staining of cytoplasmic microtubules in mature MCCs, stage 31 embryos were incubated for 2h in a 35-mm Petri dish containing 2 ml dibucaine hydrochloride (200 µM in 0.1X MBS; Fluka D0638-16). After a rapid wash in 0.1X MBS, embryos were immediately fixed by incubation in 100% methanol at -20°C for 2 days before being further processed for immunofluorescence staining.

To induce cytoplasmic microtubule depolymerization, stage 12 embryos were incubated in 2 µM Nocodazole Ready Made Solution (SML 1665, Sigma-Aldrich). This concentration was sufficient to cause the disappearance of cilia in mature MCCs, as revealed by α-Tubulin immunostaining.

## Flow and cilia beating measurements

Flow speed measurement with visible dyed microspheres (Bangs Laboratories: DSCR006, Mean diameter 5.19 µm, diluted 1/2 in MBS 0,1X) was done as previously reported (Nommick et al., 2022).

Cilia beating frequency was analyzed as previously reported (Nommick et al., 2022).

Computation of cilia beat frequency (CBF) was done using an in-house routine developed in python (Loiseau, 2020). The resulting frequencies (Hz) shown in the
quantification represent the mean CBF computed over all visible cilia per MCC. Movies are played at 50fps to best observe the beating strokes and defaults. After flow and CBF recording, MO efficiency was assessed in those embryos by immunostaining for Centrin, acetylated-Tubulin (to corroborate the presence of cilia) and mRFP (as MO injection tracer).

## Time-lapse analysis

Z-stacks from confocal video-microscopy were converted into single plane projection by maximum intensity or sum intensity projection.

All *alpha-tubulin::LifeAct-GFP*-positive MCCs were manually tracked with the 'manual tracking' Fiji plugin. To overcome the loss of focus, artefactual movements and tissue deformation events, which made the detailed analysis of cell migration and their path tracking impossible, a semi-automatic video refocusing and stabilization procedure was implemented (Chuyen et al., 2021a). For each refocused and stabilized video, MCC centroids were manually tracked using the "Manual Tracking" Fiji plugin and their position corrected using a one-dimensional Gaussian filter (sigma=10). A set of "behavioral descriptors" was then calculated to help quantify individual cell movements and their interactions throughout a whole video using a homemade Python script (Chuyen et al., 2021a). For every MCC and at every frame the euclidean distance of the centroids between two consecutive frames was calculated as well as the cumulative euclidean distance from the first frame. The instant speed was defined as the difference of the cumulative sum of the euclidean distances between the centroid positions at two consecutive time points divided by the elapsed time. These values were converted from pixel/frame to µm/min using the corresponding video acquisition
parameter. Cell contact duration was estimated by counting the time between the first and the last non-ambiguous contact time points.

Mutual contacts and overlap to ZO-1-positive vertices were manually defined. MCCs were considered intercalated when their apical surface was clearly outlined by a ZO-1 signal.

## Mathematical modelling

We simulated the system using Monte Carlo simulations (Chuyen et al., 2021b). In short, cell interactions are represented through interaction energies that the system tends to minimize.

For each MCC, a first energy term accounts for the affinity for junctions of the outer layer. This affinity energy decreases when the contact area with outer junctions increases. A second term accounts for the repulsion between MCCs. This repulsion energy increases when the distance to other MCCs decreases. The total energy of the system is the sum of the energies of all MCCs.

To implement dynamics (and to allow the system to reach energy minima), a random displacement is attributed to MCCs at each simulation time step. Possible displacements have a gaussian distribution of width σ, which effectively sets the diffusion constant of MCCs. A displacement is systematically accepted if it lowers the total energy of the system. In contrast, if the displacement increases the total energy of the system, the probability that the displacement is accepted decays exponentially with the energy jump. To account for the reduced mobility observed in *odf2* morphant embryos, σ is equal to 1/20th of a cell radius vs 1/10th in the control situation. All the other parameters are unchanged, as given in simulations of the control condition in Chuyen et al. (2021).
In both simulations and experiments, we compute the MCC order index as a measure of the spread of the areas of Delaunay tessellation triangles (Chuyen et al., 2021b), such that:

$$I = (1 - \rho) \left( 1 - \frac{sd(A)}{mean(A)} \right)$$

where A is the distribution of Delaunay areas, and ρ the cell density. Thus, the index is minimized when there is high MCC spacing variability, and maximized when there is low variability.

## Statistics

For all experiments, statistical analyses of significance were done using GraphPad Prism (version 8.2.0 for Windows, GraphPad Software, San Diego, California USA). First, the normality of data (Gaussian distribution) was tested using D'agostino & Pearson test (if n>50) or Shapiro-Wilk test (if n<50). When the data followed a normal distribution, we then compared them using parametric Student's T-tests (between 2 groups) or one One-way ANOVA (between more than 2 groups). When data did not follow a normal distribution, we compared them using Mann-Whitney (2 groups) or Kruskal-Wallis (over 2 groups) non-parametric tests. For all those tests: p=0.0001 to 0.001 or <0.0001 was considered extremely significant (***), p=0.001 to 0.01 was considered very significant (**); p=0.01 to 0.05 was considered significant (*) and p≥0.05 was considered not significant (ns). To compare the percentages between two populations we used Fisher's test. For this test p<0.12 (ns), p<0.033 (*), p<0.0021(**), p<0.0002(***) and p<0.0001(****). Number of embryos and experiments performed for all analysis are listed in table 4.
763 Acknowledgements

764 Imaging in IBDM was performed on PiCSL-FBI core facility, which is supported by the
765 French National Research Agency through the program "Investments for the
766 Future" (France-BioImaging, ANR-10-INBS-04), and is member of the Marseille
767 Imaging Institute, an Excellence Initiative of Aix Marseille University A*MIDEX, a
768 French "Investissements d'Avenir" programme (AMX 19 IET 002). The authors thank
769 Florian Roguet for Xenopus care.

770

771 Authors contribution

772 AN performed all microinjections, and all subsequent experiments related to the role
773 of Odf2 in MCC organization. AC and AN performed video-microscopy recording of
774 MCC movement. VT performed MCC spacing rescue experiments. OR generated odf2
775 expression constructs. FD wrote scripts to analyze MCC movement. RC designed and
776 performed mathematical simulations. EL performed and analyzed ciliary beating
777 experiments. FR and NB carried out EM tomography. As senior scientists in the leading
778 team, LK, CB, and OR analyzed all experiments in this study. LK and AN drafted the
779 original manuscript. All authors commented and edited the manuscript. LK designed
780 and supervised the study and obtained funding.

781

782 Competing interests

783 The authors declare no competing interests.

784
785 **Funding**

786 This project was funded by grants from the Agence Nationale de la Recherche (Oricen,

787 15-CE13-0003-02; MCCproteome 19-CE13-0033) and the Fondation pour la

788 Recherche Médicale (EQU201903007834). AN was supported by the French Ministry

789 for Research, Superior Education and Innovation, and by the Fondation pour la

790 Recherche Médicale.

791

792 **Data availability**

793 All relevant data can be found within the article and its supplementary information.

794

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900 CDC20B is required for deuterosome-mediated centriole production in multiciliated cells. Nat 901 Commun 9, 4668.

902 Ryu, H., Lee, H., Lee, J., Noh, H., Shin, M., Kumar, V., Hong, S., Kim, J., Park, S., 2021. The 903 molecular dynamics of subdistal appendages in multi-ciliated cells. Nat Commun 12, 612.

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906 Spassky, N., Meunier, A., 2017. The development and functions of multiciliated epithelia. Nat 907 Rev Mol Cell Biol 18, 423-436.

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938 multiciliogenesis. Nat Cell Biol 15, 1434-1444.

939

Figure legends

940

**Figure 1. Odf2 associates with centrioles in multiciliated cells.**

941

942 (A,B) Embryos were injected in ventral animal blastomeres at the 8-cell stage with
943 mRNA encoding Odf2-GFP (A,B) and Centrin-RFP (B), fixed at stage 18 (A), or stage
944 31 (B), and processed for immunostaining. Maximum Intensity Projections (MIPs) of
945 confocal acquisitions reveal the respective localization of Odf2-GFP (green), Centrin
946 or Centrin-RFP (red, centriole), and γ-Tubulin (white, basal foot). The bottom row
947 shows higher magnification views of images from the middle row. (C,D) MIPs of
948 confocal acquisitions showing immunostaining against Odf2 (green), and γ-Tubulin
949 (purple) at stage 18 and 31. Insets in A and C show higher magnification views on few
950 centrioles. The bottom row in B and D shows the top and lateral views of a single BB
951 analysed in 3D with Clear Volume. Scale bars: 5 µm (A-D), 1 µm (insets), 500 nm
952 (bottom row).

953

954 **Figure 2. Odf2 is necessary for basal body docking and orientation.**

955 (A) MIPs of confocal acquisitions of MCCs from uninjected control, *odf2* ATG and *odf2*
956 Spl morphant embryos at stage 31, immunostained against Centrin (red) to reveal
957 centrioles/BBs. White dotted lines indicate the plane of the Z-projections shown below.

958 (B) Graph displaying the apico-basal (A-B) distribution of centrioles/BBs within
959 uninjected control, *odf2* ATG, *odf2* Spl morphant and rescued (*odf2* ATG or Spl MO +
960 *odf2* mRNA) MCCs. Each point represents the average distance within a single cell of
961 centrioles/BBs from the most apical centriole-positive confocal slice. The middle
962 horizontal line represents the mean; the top and bottom horizontal lines represent the
963 standard deviation. (C) MIPs of confocal acquisitions of control and morphant MCCs
964 immunostained against Centrin (red) and γ-Tubulin (white, basal foot). The insets show
965 higher magnification views to best reveal BB orientation. Below each cell, a rose
966 histogram represents the circular distribution of BB orientation angles from 0 to 360°.

967 The black line running from the centre of the diagram to the outer edge is the mean
968 angle and the arcs extending to either side represent the confidence limits of the mean
969 fixed at 95% (when the mean angle is not significant, the line is pink). The length of
970 each sector corresponds to the percentage of BBs located in this range of angles. (D)
971 Graph plotting the calculated Circular Standard Deviation of BB orientation angles from
972 control, morphant and rescued MCCs. Each point represents a single cell. The middle
973 horizontal line represents the mean; the top and bottom horizontal lines represent the
974 standard deviation. (E) Graph displaying the percentage of non-polarized MCCs based
975 on the Rayleigh statistical test (MCCs with no significant mean angle of BB orientation)
976 of control, morphant, and rescued MCCs. The 5 µm scale bar in (A) applies to all
977 photographs. Scale bar in insets: 1 µm.

978

979 **Figure 3. Odf2 is required for correct basal foot assembly.**

980 (A) Transversal Transmission Electron Microscopy (TEM) acquisitions of MCCs from
981 control and *odf2* Spl morphant embryos at stage 31 were made to examine BB docking
982 at the apical membrane. Red triangles indicate BBs that failed to dock. (B) TEM
983 acquisitions of control and morphant MCCs from an apical view. Red arrows indicate
984 BB orientation vectors, based on BF direction when it is visible on the section. (C)
985 Quantification of the percentage of undocked BBs in control and morphant MCCs from
986 TEM acquisitions. (D) Quantification of BB orientation based on BF direction in control
987 and *odf2* morphant MCCs from TEM acquisitions. Each point represents the degrees
988 of deviation of a single BB, from the average angle of all BBs analysed. The middle
989 horizontal line represents the mean; the top and bottom horizontal lines represent the
990 standard deviation. (E) Quantification of BF presence in control and *odf2* morphant
991 TEM acquisitions of docked BBs, assuming that the probability of cutting at the BF
992 level is the same in both conditions. (F) Top and lateral views of 3D reconstructed and
993 colorized tomograms of control and morphant BBs. The BB is colorized in green, and
994 microtubules (MTs) in purple. In a normal BB, several MTs emanate from the BF. By
995 contrast, the Odf2-depleted BB has a smaller BF (blue arrow) with no MTs emanating
996 from it. (G) Quantification of the length (from the center of the BB cylinder to the BF
997 tip), the width (at the proximal base of the BF) and the thickness of the BF (along the
998 Z plane) of all control and morphant BBs analysed by 3D tomography. The little TEM
999 inset on the graph shows a BB with the axes considered to measure the length and
1000 the width (yellow arrows). Note that one BB entirely lacked the BF and scored 0 for all dimensions. (H) Quantification of the numbers of MTs in contact with BFs of control and morphant BBs analysed by 3D tomography.
1001
1002
1003

Figure 4. Odf2 knockdown impairs apical microtubule network assembly.

1004
(A) MIP of confocal acquisitions of a control MCC at stage 31, immunostained against
1005
Alpha-Tubulin (green, MTs) and Centrin (red). (B) MIP of confocal acquisitions of an
1006
MCC co-injected with *odf2* Spl-MO and mRNA encoding Centrin-RFP (red) to serve as
1007
injection tracer, and to reveal centrioles. In both (A) and (B) a treatment was applied
1008
to remove cilia, so as to properly visualise the cortical MT signal. (A',B') Higher
1009
magnification views of single apical confocal slices of the MCCs showed in (A,B). The
1010
regular MT network that links BBs in control cells is severely reduced in morphant
1011
conditions. The bottom row shows Z-projection views of A' and B'. The 5 µm scale bar
1012
in (A) applies to all photographs shown in (A,B). The 1 µm scale bar in (A') applies to
1013
(B').
1014
1015

Figure 5. Odf2 depletion impairs ciliary beating and fluid flow production.

1016
(A) Quantification of ciliary beating frequency (CBF/MCC in Hz) in control and
1017
morphant MCCs, based on phase-contrast microscopy and high-speed video-
1018
recording. Each dot represents the mean CBF computed over all clearly discernible
1019
cilia per MCC. Two experiments are shown, where the mean CBF of control MCCs
1020
was different, but systematically decreased upon *odf2* knockdown. (B) A pulse of red
1021
visible dye was delivered at the anterior tip of control or morphant embryos. The graph
1022
displays the percentage of the embryo length reached by the dye front in 12 seconds.
1023
Each bar represents one recorded embryo. Asterisks point the embryos showed in (C).
1024
1025 (C) Still frames at 4 time-points showing the antero-posterior progression of the red
1026 dye along the flanks of control and morphant tadpoles.

1027

1028 **Figure 6. Odf2 is necessary for correct MCC spacing.**

1029 (A,B) MIPs of confocal acquisitions of control and *odf2* morphant embryos at stage 20
1030 (before MCC intercalation, A) and stage 31 (after MCC intercalation, B). MCCs were
1031 labelled by fluorescent *in situ* hybridization (FISH) with the *alpha-tubulin* riboprobe
1032 (red) at stage 20 and by immunostaining against Centrin at stage 31 (red). ZO-1
1033 immunostaining (green) was used to reveal junctions between outer-layer cells. (A,B)
1034 Scale bar is 25 µm. (C) Graph displaying the percentage of MCCs found isolated or in
1035 contact with one or more MCCs, in control and morphant embryos. (D) Percentage of
1036 intercalated MCCs per field, in control and morphant embryos, at stage 20 and stage
1037 31. Intercalation was scored positive when an MCC apical area delimited by ZO-1
1038 staining was clearly discernible. Note that intercalation appeared delayed at stage 20
1039 in morphant embryos compared to control, but was complete at stage 31. (E) Order
1040 index of control and morphant embryos at stage 31, calculated from Delaunay
tessellation of the centroids of *alpha-tubulin*-positive MCCs. Each point represents one
1041 field of view. (F) Embryos were injected with *odf2* Spl or ATG morpholinos, with or
1042 without *odf2* mRNA, and MCC spacing was scored at st27. Note that MCC spacing
1043 was significantly rescued for both *odf2* morphant conditions.
1044

1045

1046 **Figure 7. Odf2 is necessary to maintain optimal MCC migration and homotypic repulsion.**
1047

1048 (A) Still frames of spinning disk confocal video-microscopy from Movie 5. 8-cell
1049 embryos were injected in ventral animal blastomeres with linearized plasmid DNA
1050 *alpha-tubulin::LifeAct-GFP*, to drive expression specifically in MCCs. At stage 14,
1051 embryos were processed for live imaging. Colored numbers point different MCCs to
1052 appreciate their mobility over time (hh:mm). Note that the respective position of MCCs
1053 change over time in control but not *odf2* morphant embryos. White arrows point cellular
1054 protrusions, which retract in subsequent frames. Scale bar is 25 µm. (B) Instant speed
1055 of control (n=68) and *odf2* morphant (n=62) MCCs recorded along 4h-long movies.
1056 The grey and green areas represent the standard error of all tracked MCCs. The grey
1057 and green lines represent the mean instant speed of all tracked MCCs. (C) Graph
1058 displaying the cumulative distance covered by each MCC from control and morphant
1059 embryos in 4h. (D) Graph displaying the displacement of MCCs (based on their initial
1060 and final positions) from control and morphant embryos. (E) Graph displaying the
1061 duration of MCC mutual contact in control and morphant embryos. (F) Embryos were
1062 injected with mRNA encoding mRFP to reveal the junctions between outer-layer cells.
1063 The graph displays the proportion of MCCs that migrate along outer-layer junctions
1064 (based on the overlap between GFP and RFP signals) over a period of 4h in control
1065 and morphant conditions. (G) The graph displays the order index of MCCs computed
1066 in our mathematical model over the fictive time represented by the sequential steps of
1067 the Monte-Carlo simulation. Note that when mobility is reduced the order index is stably
1068 decreased.

1069

1070 **Figure S1. Odf2 isoforms.**

1071 (A) Whole-mount *in situ* hybridization (WISH) with *odf2* riboprobe at stage 20 revealed
1072 a salt and pepper pattern typical of MCCs. (B) WISH at stage 25 combined with
1073 acetylated-Tubulin IF confirms that *odf2* transcripts are particularly enriched in MCCs.
1074 (C) Amino-acid (aa) sequences of *Xenopus Odf2* isoform 1 (short, 649aa) and *Odf2*
isoform 2 (long, 827aa). Red boxes indicate the conserved N-terminal aa-sequence similar of the human Cenexin insertion. In blue are shown the C-terminal short and long sequences that differ between the two isoforms. The region expected to be recognized by the human ODF2 antibody used in this study is underlined in blue. (D) Alignment of human CENEXIN1 and *Xenopus* Odf2 isoforms 1 and 2. In the consensus line, the amino-acids that are identical or similar between the two sequences appear in red, and those that differ appear in blue. Dots point residues that are absent in one or the other protein. Note that isoform 1 was used to generate the Odf2-GFP construct used in this study. (E) MIPs of confocal acquisitions showing Odf2 (green), and γ-Tubulin (purple, centrioles) immunostaining, at centrosomes of non-MCCs at stage 31. (F) MIPs of confocal acquisitions of immunostaining against Centrin (red, centrioles), Odf2-GFP expression (green) and γ-Tubulin (white) at centrosomes of non-MCCs at stage 31. (E,F) Scale bar is 1 µm.

1088

1089 **Figure S2. Characterization of *odf2* morpholino antisense oligonucleotides.**

(A) Schematic representation of *Xenopus laevis* *odf2* (Xenbase entrez-gene 496006).

Red bars below exon1 show the position of *odf2* ATG and *odf2* Spl MOs. In the red dashed box is shown a zoom on exon1-intron1-exon2 region with the position of the primers used to reveal intron1 retention caused by *odf2* Spl MO.

(B) Validation of *odf2* ATG MO by immunostaining. MIPs of confocal acquisitions showing two MCCs, one co-injected with *odf2* ATG MO + Centrin-RFP (white asterisk) and the other without

MO. (C) PCR on genomic DNA (left gel) extracted from *Xenopus* embryo and A6 cells amplify a fragment of 260bp containing intron1. PCR on cDNA (middle gel) prepared

from total RNA extracted at different embryonic stages reveal a 167bp fragment resulting from intron1 splicing. PCR on *Xenopus* cDNA from control and *odf2* morphant
1100 embryos at stage 20 (right gel). The presence of the 260bp band in morphant cDNA
1101 confirms intron1 retention. (D,E) For rescue experiments, embryos were sequentially
1102 co-injected at the 8-cell stage with *odf2* Spl MO, or *odf2* ATG MO and *centrin-RFP*
1103 mRNA, and at the 16-cell stage with *odf2-GFP* mRNA. Micrographs show MIPs of
1104 confocal acquisitions of MCCs at stage 31 immunostained for γ-Tubulin (BF, white)
1105 and Centrin-RFP (BB, red). Asterisks point MCCs rescued by the presence of Odf2-
1106 GFP. The white dashed lines indicate the plane of the Z views shown below. (B,D,E)
1107 Scale bar is 25 µm.
1108

1109

Figure S3. Odf2 is not required for ciliogenesis or rootlet formation.

1109

1110 (A) MIPs of confocal acquisitions of *odf2* Spl morphant MCCs at stage 31,
1111 immunostained for centrioles (Centrin, red), cilia (Acetylated-Tubulin, green) and
1112 morpholino tracer (mRFP coinjected with MO, white). **Uninjected control and odf2**
1113 morphant MCCs are identified with white and pink asterisks, respectively. **Scale bar is**
1114 50 µm. Dashed boxes delimit the regions magnified in A' and A". In A" the top
1115 morphant MCC displayed moderate BB clustering and made some cilia, whereas the
1116 bottom MCC (pink arrow) displayed severe BB clustering deep in the cytoplasm and
1117 made no cilia. The white dashed line indicates the plane of the Z view shown below.
1118 **Scale bar is 10 µm.** (B) MIPs of confocal acquisitions of control and *odf2* morphant
1119 MCCs immunostained for the rootlet marker Pcnt (green) and Centrin (red). **Scale bar**
1120 is 5 µm. Dashed boxes delimit the region magnified in the views below. **Scale bar** in
1121 the zoomed views is 1 µm. The white dashed line indicates the plane of the Z view
1122 shown below. (C) Quantification of Pcnt mean signal intensity. Each dot represents an
1123 individual MCC. The middle horizontal line represents the mean, the top and bottom
1124 horizontal lines represent the standard deviation from the mean.

1110

1111

1112

1113

1114

1115

1116

1117

1118

1119

1120

1121

1122

1123

1124
1125

1126

**Figure S4. Depletion of Odf2 impairs actin and intermediate filament networks in MCCs.**

1127

1128

(A,B) MIPs of confocal acquisitions of control and *odf2* morphant MCCs at stage 31, stained for F-actin (Sir-actin, white) and Centrin (red, centrioles). Dashed boxes indicate high magnification views in A', B'. (A',B') Single slices of confocal acquisitions showing the apical or subapical (0.6 µm below) actin network in control and morphant MCCs. We observe a strong actin signal reduction in morphant cells. The dashed white line sets the position of the corresponding Z views shown on the bottom row. (C,D) MIPs of confocal acquisitions of control and morphant MCCs at stage 31, immunostained for intermediate filaments (IFs, C-11, green) and Centrin or Centrin-RFP (centrioles, red) coinjected with *odf2* Spl-MO. Dashed boxes indicate high magnification views in C', D'. (C',D') High magnification views showing the regular IF network that surrounds BBs in control cells, which appears strongly disorganized in morphant conditions. White dashed lines set the position of the corresponding Z views shown on the bottom row. Scale bars are the same for control and morphant images. (A-D) Scale bar is 5 µm. (A'-D') Scale bar is 1 µm.

1129

1130

1131

1132

1133

1134

1135

1136

1137

1138

1139

1140

1141

1142

1143

**Figure S5. Cell-autonomous requirement for Odf2 in MCC spacing pattern establishment.** (A) MIPs of confocal acquisitions of control and *odf2* morphant embryos at stage 20, before MCC intercalation is complete. MCCs were labelled by fluorescent *in situ* hybridization (FISH) with the *alpha-tubulin* riboprobe (white). *mRFP* mRNA was co-injected with *odf2* Spl MO. Junctions between outer-layer cells were revealed by ZO-1 immunostaining (green). The dashed line in each view delimits domains where injection targeted primarily the surface cellular layer (right), or the

1144

1145

1146

1147

1148

1149
1150 internal cellular layer (left). The dashed boxes 1 and 2 point the areas magnified below.
1151 In box 1, MCCs likely received little or no *odf2* MO, as mRFP was mostly visible in
1152 superficial cells, and spaced properly. In box 2, MCCs likely received significant
1153 amounts or *odf2* MO, as they were positive for mRFP, and did not space properly.
1154 Dashed horizontal white lines indicate the position of the Z-projections shown on the
1155 bottom row. Note the superficial position of mRFP in box 1 and its deep position in box
1156 2. Scale bar is 100 µm. Scale bar in the zoomed views is 25 µm.

1157

1158 **Figure S6. MCC spacing depends on proper microtubule dynamics.** (A) Control
1159 uninjected and *odf2* Spl morphant embryos were immunostained at stage 18 to reveal
1160 cytoplasmic microtubules (α-Tubulin, green) and BBs in MCCs (Centrin or Centrin-
1161 RFP, red). Scale bar is 25 µm. (B) Graph showing the net mean α-Tubulin signal
1162 intensity in control and morphant MCCs. (C,D) Embryos were incubated in 2 µM
1163 nocodazole from stage 12 to 18 (C) and processed for *in situ* hybridization, or from
1164 stage 12 to 27 and processed for immunostaining (D), to reveal MCCs. Scale bars in
1165 (C) and (D) are 50 µm and 25 µm, respectively. (E) Graph displaying the percentage
1166 of MCCs found isolated or in contact with one or more MCCs, in control and
1167 nocodazole-treated embryos at stage 27.

1168

1169 **Figure S7. Initial and final frames from the simulation Movie 6.** The grey meshwork
1170 represents outer-layer intercellular junctions, and red circles represent virtual MCCs.
1171 The values of the order index (OI) are shown.

1172

1173
1174

<table><caption>**Table 1. Plasmids**</caption><thead><tr><td>mRNA</td><td>Quantity injected per blastomere (pg)</td><td>Stage of injection</td><td>Source</td></tr></thead><tbody><tr><td>pCS2+mRFP</td><td>200</td><td>4/8 cells</td><td>(Stubbs et al., 2006)</td></tr><tr><td>pCS2+Centrin-RFP</td><td>50</td><td>4/8 cells</td><td>(Stubbs et al., 2006)</td></tr><tr><td>pCS2+GFP-GPI</td><td>500</td><td>4/8 cells</td><td>(Stubbs et al., 2006)</td></tr><tr><td>pCS2+Odf2-GFP</td><td>100-200</td><td>8/16 cells</td><td>This study</td></tr><tr><td>pCS2+alpha-tubulin::LifeAct-GFP</td><td>50</td><td>8/16 cells</td><td>(Chuyen et al., 2021b)</td></tr></tbody></table>

1175

1176

<table><caption>**Table 2. Primary antibodies and dyes**</caption><thead><tr><td>Working fixation</td><td>Target</td><td>Reference</td><td>Dilution</td></tr></thead><tbody><tr><td>MetoH 100%</td><td>γ-tubulin mouse IgG1</td><td>Abcam Ab11316</td><td>1/800</td></tr><tr><td>4°C OW or PFA 4% 0,1%</td><td>Acetylated-tubulin mouse IgG2b</td><td>Sigma-Aldrich T7451</td><td>1/1000</td></tr><tr><td rowspan="3">Triton 30min RT</td><td>GFP chicken</td><td>AvesLab GFP-1020</td><td>1/500</td></tr><tr><td>RFP rat IgG2a</td><td>Chromotek 5F8</td><td>1/500</td></tr><tr><td>Centrin mouse IgG2a</td><td>Sigma-Aldrich 04-1624</td><td>1/1000</td></tr></tbody></table>
<table><tr><td></td><td>Cytokeratin (Pan-reactive) mouse monoclonal IgG1</td><td>Sigma-Aldrich SAB4700666</td><td>1/500</td></tr><tr><td></td><td>ZO-1 mouse IgG1</td><td>Thermo Scientific 33-9100</td><td>1/200</td></tr><tr><td></td><td>γ-tubulin mouse IgG1</td><td>Sigma-Aldrich T9026</td><td>1/500</td></tr><tr><td>Memfa 1h RT</td><td>Sir-Actin</td><td>Spirochrome SC001</td><td>1/1000</td></tr><tr><td>Memfa 2h RT or ON 4°C</td><td>Anti DIG-PA conjugated IgG</td><td>Roche 11266026</td><td>1/500</td></tr><tr><td>PFA 4% Triton 0,1x 30min RT</td><td>Pericentrin rabbit</td><td>Home Made, this study</td><td>1/5000</td></tr><tr><td>MetoH 100% 4°C OW</td><td>Cenexin1/Odf2 rabbit IgG</td><td>Abcam Ab43840</td><td>1/300</td></tr></table>

1177

1178

<table><caption>**Table 3. Secondary antibodies**</caption><thead><tr><td>Target</td><td>Reference</td><td>Dilution</td></tr></thead><tbody><tr><td>Alexa anti mouse IgG2a 488</td><td>Invitrogen Thermo Fisher A21131</td><td>1/800</td></tr><tr><td>Alexa anti mouse IgG2a 568</td><td>Invitrogen Thermo Fisher A21134</td><td>1/800</td></tr><tr><td>Alexa anti mouse IgG2a 647</td><td>Invitrogen Thermo Fisher A21241</td><td>1/800</td></tr><tr><td>Alexa anti mouse IgG1 488</td><td>Invitrogen Thermo Fisher A21121</td><td>1/800</td></tr><tr><td>Alexa anti mouse IgG1 568</td><td>Invitrogen Thermo Fisher A21124</td><td>1/800</td></tr><tr><td>Alexa anti mouse IgG1 647</td><td>Invitrogen Thermo Fisher A21240</td><td>1/800</td></tr><tr><td>Alexa anti mouse IgG2b 488</td><td>Invitrogen Thermo Fisher A21141</td><td>1/800</td></tr><tr><td>Alexa anti mouse IgG2b 568</td><td>Invitrogen Thermo Fisher A21144</td><td>1/800</td></tr></tbody></table>
<table><tr><td>Alexa anti mouse IgG2b 647</td><td>Invitrogen Thermo Fisher A21242</td><td>1/800</td></tr><tr><td>Anti rat Cy3 IgG</td><td>Jackson ImmunoResearch 712165153</td><td>1/500</td></tr><tr><td>Alexa fluor Anti rat 488 IgG</td><td>Life Technologies A21208</td><td>1/500</td></tr><tr><td>Alexa fluor Anti rat 647 IgG</td><td>Jackson ImmunoResearch 712605153</td><td>1/500</td></tr><tr><td>Alexa fluor Anti rabbit 488 IgG</td><td>Life Technologies A21206</td><td>1/500</td></tr><tr><td>Alexa fluor Anti rabbit 568 IgG</td><td>Life Technologies A10042</td><td>1/500</td></tr><tr><td>Alexa fluor Anti rabbit 647 IgG</td><td>Life Technologies A31573</td><td>1/500</td></tr><tr><td>Alexa fluor Anti chicken 488 IgY</td><td>Jackson ImmunoResearch 703545155</td><td>1/500</td></tr></table>

1179

1180

<table><caption>**Table 4. Experiment Sampling**</caption><thead><tr><th>Experiment</th><th>Condition</th><th>N° of MCCs<br>(or BBs for<br>TEM)<br>analyzed</th><th>N° of<br>embryos<br>analysed</th><th>N° of<br>experiments</th></tr></thead><tbody><tr><td rowspan="4">BB orientation<br>(and BB)</td><td>Control</td><td>164</td><td rowspan="4">3-5</td><td rowspan="4">1-2</td></tr><tr><td>odf2 ATG-MO</td><td>41</td></tr><tr><td>odf2 Spl-MO</td><td>38</td></tr><tr><td>odf2 ATG-MO<br>+mRNA</td><td>59</td></tr></tbody></table>
<table><tbody><tr><td>orientation<br>Rescue)</td><td>odf2 Spl-MO<br>+mRNA</td><td>33</td><td></td><td></td></tr><tr><td rowspan="5">MCC<br>polarization<br>(Rayleigh Test)</td><td>Control</td><td>521</td><td>9-14</td><td>3-5</td></tr><tr><td>odf2 ATG-MO</td><td>346</td><td></td><td></td></tr><tr><td>odf2 Spl-MO</td><td>113</td><td></td><td></td></tr><tr><td>odf2 ATG-MO +<br>mRNA</td><td>60</td><td>3-5</td><td>2</td></tr><tr><td>odf2 Spl-MO +<br>mRNA</td><td>33</td><td></td><td></td></tr><tr><td rowspan="3">MCC<br>polarization<br>(Rayleigh Test)</td><td>Control</td><td>521</td><td>9-14</td><td>3-5</td></tr><tr><td>odf2 ATG-MO</td><td>291</td><td></td><td></td></tr><tr><td>odf2 Spl-MO</td><td>64</td><td></td><td></td></tr><tr><td rowspan="5">BB docking</td><td>Control</td><td>28</td><td>3-5</td><td>2</td></tr><tr><td>odf2 ATG-MO</td><td>19</td><td></td><td></td></tr><tr><td>Odf2 Spl-MO</td><td>19</td><td></td><td></td></tr><tr><td>odf2 ATG-MO<br>+mRNA</td><td>19</td><td></td><td></td></tr><tr><td>odf2 Spl-MO<br>+mRNA</td><td>19</td><td></td><td></td></tr><tr><td rowspan="2">BB docking<br>(TEM)</td><td>Control</td><td>39</td><td>1</td><td>1</td></tr><tr><td>odf2 Spl-MO</td><td>35</td><td>1</td><td></td></tr><tr><td rowspan="2">BB orientation<br>(TEM)</td><td>Control</td><td>30</td><td>1</td><td></td></tr><tr><td>odf2 Spl-MO</td><td>66</td><td>3</td><td></td></tr><tr><td></td><td>Control</td><td>136</td><td>1</td><td></td></tr></tbody></table>
<table><tbody><tr><td>BF presence<br>(TEM)</td><td>odf2 Spl-MO</td><td>118</td><td>3</td><td></td></tr><tr><td rowspan="3">BF presence</td><td>Control</td><td>11</td><td rowspan="3">3</td><td rowspan="3">1</td></tr><tr><td>odf2 ATG-MO</td><td>10</td></tr><tr><td>odf2 Spl-MO</td><td>31</td></tr><tr><td rowspan="2">BB tomography</td><td>Control</td><td>2</td><td rowspan="2">2</td><td rowspan="2">1</td></tr><tr><td>odf2 Spl-MO</td><td>4</td></tr><tr><td rowspan="2">Pericentrin<br>signal</td><td>Control</td><td>24</td><td>1</td><td rowspan="2">1</td></tr><tr><td>odf2 ATG-MO</td><td>15</td><td>1</td></tr><tr><td rowspan="4">Actin signal</td><td>Control (of odf2<br>ATG-MO)</td><td>72</td><td rowspan="4">3</td><td rowspan="4">1</td></tr><tr><td>Control (of odf2<br>Spl-MO)</td><td>50</td></tr><tr><td>odf2 ATG-MO</td><td>94</td></tr><tr><td>odf2 Spl-MO</td><td>39</td></tr><tr><td rowspan="2">α-Tubulin signal<br>quantification</td><td>Control</td><td>113</td><td rowspan="2">5</td><td rowspan="2">1</td></tr><tr><td>odf2 Spl-MO</td><td>69</td></tr><tr><td rowspan="2">Flow<br>engagement</td><td>Control</td><td></td><td rowspan="2">12</td><td rowspan="2">2</td></tr><tr><td>odf2 Spl-MO</td><td></td></tr><tr><td rowspan="2">Cilia beating</td><td>Control</td><td>38</td><td rowspan="2">15</td><td rowspan="2">2</td></tr><tr><td>odf2 Spl-MO</td><td>32</td></tr><tr><td rowspan="2">MCC contacts</td><td>Control St 20</td><td>1692</td><td>7</td><td rowspan="2">2-3</td></tr><tr><td>Control St 31</td><td>1944</td><td>12</td></tr></tbody></table>
<table><tbody><tr><td></td><td>odf2 Spl-MO St<br>20</td><td>1605</td><td>7</td><td></td></tr><tr><td></td><td>odf2 Spl-MO St<br>31</td><td>2676</td><td>26</td><td></td></tr><tr><td></td><td>mRFP St 20</td><td>653</td><td>4</td><td></td></tr><tr><td></td><td>mRFP St 31</td><td>474</td><td>4</td><td></td></tr><tr><td>MCC contacts</td><td>Control</td><td>259</td><td>6</td><td>1</td></tr><tr><td>Nocodazole</td><td>NOC 2µM</td><td>226</td><td>8</td><td></td></tr><tr><td rowspan="2">MCC<br>displacement<br>and cumulative<br>distance</td><td>Control</td><td>69</td><td>4</td><td>4</td></tr><tr><td>odf2 Spl-MO</td><td>59</td><td>2</td><td>2</td></tr><tr><td rowspan="2">MCC time of<br>contact and<br>association to<br>junction</td><td>Control</td><td>102</td><td>5</td><td>5</td></tr><tr><td>odf2 Spl-MO</td><td>86</td><td></td><td></td></tr><tr><td rowspan="4">MCC<br>intercalation</td><td>Control St 20</td><td>604</td><td>4</td><td>2</td></tr><tr><td>Control St 31</td><td>522</td><td>5</td><td>3</td></tr><tr><td>odf2 Spl-MO St<br>20</td><td>830</td><td>5</td><td>2</td></tr><tr><td>odf2 Spl-MO St<br>31</td><td>552</td><td>5</td><td>3</td></tr><tr><td>MCC spacing</td><td>mRFP</td><td>1217</td><td>9</td><td>1</td></tr><tr><td rowspan="2">Rescue</td><td>Odf2-GFP</td><td>1004</td><td>6</td><td></td></tr><tr><td>odf2 ATG-MO</td><td>2086</td><td>8</td><td></td></tr></tbody></table>
<table><tr><td></td><td><u>odf2 Spl-MO</u></td><td><u>1391</u></td><td><u>6</u></td></tr><tr><td></td><td><u>odf2 ATG-MO + mRNA</u></td><td><u>2136</u></td><td><u>14</u></td></tr><tr><td></td><td><u>odf2 Spl-MO + mRNA</u></td><td><u>1951</u></td><td><u>13</u></td></tr></table>

1181

1182

1183 Movies

1184 1. 3D tomography-reconstruction of a mature control basal body (related to Fig. 3F)

1185 2. 3D tomography reconstruction of an *odf2* Spl morphant basal body (related to Fig. 3F)

1186 3. Ciliary beating of control and *odf2* Spl morphant MCCs (related to Fig. 5A)

1187 4. Flow dynamics along the flanks of control and *odf2* Spl morphant tadpoles (related to Fig. 5C)

1188 5. Cell motility of control and *odf2* Spl morphant MCCs at stage 14 (related to Fig.7A)

1189 6. Mathematical simulation of pattern establishment with reduced MCC motility (related to Fig.7G and S5B).

1194