Fig. 5: Depletion of FMRP promotes degeneration of ependymal multicilia.
From: Cilia locally synthesize proteins to sustain their ultrastructure and functions
Pooled data were from three independent experiments and are presented as mean ± s.d. Two-tailed student’s t-test against control populations: ns, no significance. a Experimental scheme. mEPCs transfected with siRNA (Ctrl-i or Fmr-i1) were harvested at day 6 or 12 to assess the status of multiciliation. b Fmrp was dispensable for multiciliated cell formation. 1280 (Ctrl-i, Day 6), 1456 (Fmr-i2, Day 6), 1501 (Fmr-i1, Day 6), 1350 (Ctrl-i, Day 12), 1225 (Fmr-i2, Day 12), and 1360 (Fmr-i1, Day 12) cells from three biologically independent experiments were scored. c Representative patterns of multicilia in day-12 mEPCs transfected with Fmr-i1. Odf2 and Cep164 served as basal body markers. Nascent: short multicilia with scattered basal bodies; Grown: long multicilia with packed basal bodies; Degenerating: a few uneven multicilia with overwhelming numbers of tightly packed basal bodies. d Depletion of Fmrp increased mEPCs with degenerating multicilia at day 12. mEPCs were scored following the criteria in c. 635 (Ctrl-i, Day 6), 789 (Fmr-i2, Day 6), 895 (Fmr-i1, Day 6), 883 (Ctrl-i, Day 12), 690 (Fmr-i2, Day 12), and 899 (Fmr-i1, Day 12) cells from three biologically independent experiments were scored. e Experimental scheme for tracing dynamic changes of multicilia through live imaging. mEPCs were infected with adenovirus to express Centrin1-GFP, followed by siRNA (Ctrl-i or Fmr-i1) transfection. Spinning disk microscopy was performed from day 7 for 24 h at 10 min intervals after the addition of siR-tub to label axonemes. At each timepoint, z-stack images were acquired at 0.5 μm intervals for a depth of 15 μm to sufficiently cover basal bodies and ciliary tips. f Representative frames showing multicilia degeneration in mEPCs transfected with Fmr-i1. Both z-projected images (x-y) and their orthogonal views of 3D-reconstructed images (x-z) are presented. The dashed line marks the middle plane of basal bodies. Time stamps are in the format of hours: minutes. g Quantification of 60 multiciliated cells in each condition. Multicilia undergoing degeneration are marked in blue.
