Fig 2: Cdk5rap2null erythroblasts undergo fewer divisions and enucleate prematurely Deconvolved STED images of centrosomes in ex vivo cultured wild-type non-enucleating EBs (non-enucl.) or enucleating EBs/reticulocytes (enucl./RetiC). Cells were stained for ?-tubulin (magenta) and centriolar (CP110, CEP135) or PCM (CDK5RAP2, PCNT) proteins (cyan). Scale bar, 400 nm.Quantification of number of cell divisions after 24 (T24) and 48 (T48) hours. Ex vivo cultured erythroid progenitors with indicated genotypes were labeled with PKH26 to measure cell divisions. The numbers in brackets refer to the number of embryos analyzed.Schematic showing how ex vivo cultured erythroid progenitors were tracked in bright-field time-lapse microscopy experiments. Briefly, the two daughter cells produced by the first cytokinesis and their progeny were followed through terminal erythroid differentiation. Whenever a cell performed cytokinesis, enucleation, or death, this was recorded as an event (ev). Examples depict paths taken by four daughter cells.Quantification of the frequency of cytokinesis, enucleation, and death at each event (ev) in Cdk5rap2 wild-type or null erythroid progenitors as shown in (C). Four embryos for each genotype were analyzed.Quantification of cell cycle duration of Cdk5rap2 wild-type or null erythroid progenitors. The enucleation event was used as reference point to align previous divisions (see schematic in C). The number in brackets refers to the number of embryos analyzed. Data information: Box plots show 5th and 95th (whiskers) and 25th, 50th, and 75th percentiles (boxes). Bar graphs display mean ± s.d. All statistical analysis was based on the number of embryos. Statistical significance was determined by one-way ANOVA with Tukey’s multiple comparisons test (B) or Mann–Whitney U-test (D and E). *P = 0.05, **P = 0.01.

Fig 3: Knockdown of CP110 reverses the ciliary defects in NudCL2 morphants.Embryos were injected with the indicated MOs and harvested at different times. a Immunoblot analysis of NudCL2 and CP110 expression. a-tubulin, a loading control. b–d Immunofluorescence of cilia with anti-acetylated-a-tubulin antibody in KVs at 6–8 somite stages (b). Scale bar, 10 µm. The cilia number per KV was calculated (c). Cilia length in KV was measured using ImageJ software (d). e–h Whole-mount in situ hybridization with cmlc2 probe (48 hpf) and foxa3 probe (72 hpf). Liver and pancreas are indicated by arrows and arrowheads, respectively. The percentage of the expression patterns of cmlc2 and foxa3 was calculated (g, h). The data are presented as means ± SD derived from at least three independent experiments. n, sample size. **P < 0.01 and ***P < 0.001 Student’s t-test. i Working model of NudCL2-mediated autophagic degradation of CP110 at mother centrioles to initiate ciliogenesis. See text for details.

Fig 4: PACSIN1 functions at the CV stage prior to CP110 loss. a Quantification of CP110 dots in serum starved RPE-1 cells treated with siRNAs for 72 h, serum starved for the last 24 h, and stained with CP110, Actub and CEP164 antibodies. CP110 localization on both centrioles (2 dots) or only the daughter centriole (1 dot) was quantified (left panel, siCtrl = 100, siPACS1 = 63 cells). b Quantification of CEP97 dots in cells treated as in a and stained with CEP97, Actub, and CEP164 antibodies. CEP97 localization on the centrioles was quantified as in a(siCtrl = 201, siPACS1 = 125 cells). c Quantification of SMOM2-GFP centrosomal accumulation in cells treated as in a, and stained with Actub and CEP164 antibodies. “Preciliary” indicates SMOM2-GFP vesicle accumulation at the MC without axonemes (siCtrl = 100, siPACS1 = 67 cells). Scale bar: 2 µm for images in a–c. d Quantification of MC with non-ciliary distal appendages structures from cells treated as in a. Pooled data from 3 independent experiments (siCtrl = 56, siPACS1 = 41 cells). Representative electron micrographs of quantified MCs shown on right. Scale bar: 200 nm. MC mother centriole, DAV distal appendages vesicles, CV ciliary vesicle, or DA non-membrane-associated distal appendages. e–g Quantification of TZ accumulation of TMEM67 (e, siCtrl = 113, siPACS1 = 89 cells), B9D2-GFP (f, siCtrl = 365, siPACS1 = 250 cells), RPGRIP1L (g, siCtrl = 70, siPACS1 = 63 cells) in cells treated as in a. h mIFT20-GFP cells treated as in a, stained with Actub and CEP164 antibodies, and quantified for mIFT20 fluorescence intensity at the MC/BB (siCtrl = 46, siPACS1 = 43 cells). Scale bar: 2 µm for images in e–h. In a–c, and e–h, representative images of quantified cells shown on right and means ± SEM, n = 3 independent experiments. Two-tailed t-test analysis as compared with siCtrl was performed. **P < 0.001, ***P < 0.0001

Fig 5: The POC16 Human Homolog WDR90 Is Required for Ciliogenesis(A) Confocal images of RPE-1 p53- cells after induction of ciliogenesis, stained for MonoE (cyan) and WDR90 (red); DNA is visible in blue throughout this figure. Note that WDR90 marks both mother and daughter centrioles. The scale bar represents 2 µm, and all panels are at the same magnification.(B–D) Insets of human centrioles from RPE-1 p53- cells stained with antibodies against MonoE (cyan) and CP110 (magenta) (B), MonoE (cyan) and WDR90 (red) (C), or Centrin (green) and WDR90 (red) (D), as indicated.(E) Distances from the centers of the CP110 signal to the MonoE signal (280 ± 52 nm), as well as from the WDR90 signal to the MonoE signal (177 ± 56 nm) or to the Centrin signal (61 ± 46 nm). Schematic representation of the results is shown in the upper inset. Data are represented as mean ± SD.(F and G) Confocal images of RPE-1 p53- cells transfected with control siRNAs (F) or with siRNAs targeting WDR90 (G), serum-starved for 48 hr to induce ciliogenesis and then analyzed by immunofluorescence against BBS4 (red) and MonoE (green). The scale bar represents 2 µm.(H) Percentage of RPE-1 p53- cells harboring a primary cilium in the indicated conditions 48 hr after serum starvation. Average for control ± SD: 68% ± 10%; siWDR90: 32% ± 11%. Student two-tailed t tests are as follows: control/siWDR90; p = 0.01608.(I and J) Confocal images of RPE-1 p53- cells transfected with control siRNAs (I) or siRNAs targeting WDR90 (J), serum starved for 48 hr to induce ciliogenesis, and then analyzed by immunofluorescence against WDR90 (red) and MonoE (green). The scale bars represent 2 µm.(K) Confocal images of RPE-1 p53- cells transfected with siRNAs targeting WDR90, induced by doxycycline to express RNAi-resistant WDR90, serum starved for 48 hr to induce ciliogenesis, and then analyzed by immunofluorescence against WDR90 (red) and MonoE (green). The scale bar represents 2 µm.(L) Percentage of RPE-1 p53- cells harboring a primary cilium in the indicated conditions 48 hr after serum starvation. Average for control ± SD: 62% ± 5%; siWDR90: 27% ± 7%; siWDR90+WDR90RR: 48% ± 7%. Student two-tailed t tests are as follows: control/siWDR90, p = 0.00241; siWDR90/siWDR90+WDR90RR, p = 0.02441.(M) Schematic representation of Chlamydomonas and human centrioles, highlighting the central core region (red) located in the middle of the Chlamydomonas centriole—hence its name—and in the human centriole. POB15 and POC16/WDR90 both reside in the inner barrel within these regions.See also Figures S6 and S7.