Fig. 7: BB polarities and AOs are initially decoupled during mouse development.

a Wild-type P4 ependymal tissues were abundant in multicilia with uncoordinated beats (asterisks). Ciliary motilities were imaged at 10 ms intervals. The first three consecutive frames were pseudo-colored and merged to show ciliary motilities. Arrows indicate beat directions of multicilia showing coordinated beats. Refer to Supplementary Movies 7 and 8. b Workflow for AutoCUTS-SEM. Ependymal samples were fixed, embedded in resin, and serially sectioned using AutoCUTS. Ultrathin sections were adhered to tape and transferred onto a wafer. High-throughput SEM images were acquired⁴⁵, especially in regions with diverse AOs. c, d P4 ependymal cells contained multicilia with diverse AOs. AOs are indicated by arrows in the typical SEM image (c), and their distributions (d) were quantified from six SEM micrographs, each containing at least 13 clear axonemal cross-sections. e, f BF-AO relationships in P4 cilia. Two numbered cilia were tracked from axonemes to BFs in representative serial cross-sections from Supplementary Movie 9 (e). Radar charts (f) show included angles between the AO and BB polarity from 41 cilia in 4 cells. The red and blue dots were from regions with diverse and uniform AOs, respectively. An unpaired two-tailed student’s t-test was performed. g Model for progression of ciliary rotational polarities. Cilia are depicted as top views to show AOs (arrows) and BFs (green). For simplicity, the translational polarity is not depicted. Nascent multicilia initially lack BF-AO coupling and display uncoordinated beat due to randomized AOs (phase I). Axonemes in individual cells gradually unify their AOs to achieve the rotational polarity and coordinated beats (phase II), with some possibly achieving BF-AO coupling. In phase III, BF-AO coupling and rotational polarity of BBs are achieved in both individual and different E1 cells, leading to tissue-level directional beats of multicilia. BB-localized Ccdc57 shifts from a random position relative to the BF (purple spot) in phase I to a position opposite the BF (red spot) in phase III to fix the BF-AO relationship. Cultured mEPCs only reach phase II naturally, whereas E1 cells in the ependyma progress to phase III at around P21. Source data are provided as a Source Data file.