Primary Cilia

A Leica SP2 confocal microscope was used to create maximum projections of confocal z-stacks (Fig. 1a) from which cilia length was measured using image J software. At least three different mounted preparations were used to capture five fields of cells at ×63 magnification giving data for >100 cilia per subgroup. Confocal z maximum projections were also used to assess cilia prevalence and ki-67 nuclear staining.Fig. 1

IL-1 increases primary cilia length in chondrocytes. a Chondrocyte primary cilia (green, anti-acetylated-α-tubulin, blue DAPI nuclei). Scale bar 10 μm. b Frequency histogram of chondrocyte cilia length showing elongation of cilia associated with IL-1β treatment (10 ng mL⁻¹, 24 h). Insert shows that negligible cells were in a proliferative state as indicated by nuclear ki-67 expression. c IL-1β at 2–50 ng mL⁻¹ induced statistically significant increases in cilia length as did IL-1α at 10 ng mL⁻¹, but no statistically significant differences were seen between the different concentration or type of IL-1 used. d The influence of IL-1 was observed at 3 h and the effect maintained throughout a 48-h treatment period. Statistically significant differences are indicated relative to untreated control

For the detection of primary cilia in vitro, cells were maintained in serum-starved culture medium for 24–72 h^{31 (link)–34 (link)}. Briefly, HeLa cells were grown on coverslip and then incubated with serum-starved DMEM with 0.1% FBS for 36 h. Cells were fixed with 4% paraformaldehyde for 10 min, washed three times with cold PBS, and permeabilized with PBST (0.1% (v/v) Triton X-100 in PBS) for 10 min. Then, cells were washed three times, and incubated with monoclonal anti-acetylated tubulin antibodies diluted (1:1000) in PBST for 1 h at room temperature. After washing three times with PBS, cells were incubated with FITC-conjugated goat anti-mouse IgG-secondary antibody or goat anti-mouse IgG-Alexa 568 diluted (1:1000) in PBST for 1 h at room temperature. Nucleus was visualized by staining cells with DAPI. After washing with PBS, cells were mounted on glass slide. Primary cilia were observed and photographed at 1,000 x magnification under a fluorescence microscope (Nikon, Tokyo, Japan).

Genes biologically related to cilia were obtained from SYScilia database [67 (link)] (https://.Syscilia database.com). These genes were separated into two subsets of genes regarding their involvement in either primary or in motile cilia (Supplementary Table 5). The coverage of the selected genes in the exome was above 70% (exon_coverage_20x) except for SHH with a respective coverage of 59%. As control genes for permutations tests, 1,926 housekeeping genes identified in at least seven different studies (detective breadth ≥ 7; [107 (link)]) were used.
WES data from 25 unrelated hydrocephalus patients and 166 in-house controls, composed of patients presenting pathologies other than cerebral and their relatives, were used for the genetic mutation burden test analysis.
Population stratification of unrelated hydrocephalus patients and control patients was determined by principal component analysis (PCA) using PLINK software [108 (link)]. Mahalanobis distance (MD) with a significance level of 5% was used to identify potential outliers.
A genetic mutation burden test was used to assess if there was a significant excess of variants in ciliary genes in our patients compared to controls. WES data of controls and cases were analyzed to search for variants in genes related to ciliary structure. Variants were filtered for quality criteria (pass GATK [102 (link)] standard filter, read depth ≥ 10), AF (< 30%, 10%, 5%, 3%, 1%, 0.5% based on the maximum minor allele frequency found in ExAC [36 (link)], 1000G [37 (link)], ESP650 [39 ], GoNL [38 ], ARIC5606 [104 ], and our in‐house database) and mutation impact using snpeff_effect [106 (link)].
The genetic burden was analyzed using an in-house developed program in Python (https://www.python.org/). Statistical significance was measured by comparing the genetic burden of our patients to controls using a nonparametric Wilcoxon test. Precisely, for each patient or control, the number of allelic variants in ciliary genes was counted, with homozygous variants counting as two allelic variants. A permutation test with 10,000 random selections of 304 or 253 housekeeping genes was performed to exclude the effect of chance in all/primary cilia gene selection, respectively. A mutation burden was measured and the Wilcoxon statistic for independent samples was calculated for each of the 10,000 selections. The number of subsets of housekeeping genes, yielding a Wilcoxon statistic with a smaller p value than for the ciliary genes, was counted and divided by 10,000. This value was set as the p value for the permutation tests. An explanatory scheme is available in Additional file 1: Fig. S1.

Matched Z stack maximum projections were analyzed in Fiji. Relative ciliary enrichment was calculated as follows: each primary cilium was manually defined by a segmented line following ADCY3⁺ (in vivo) or ACTB⁺ (in vitro) signal. This 2D space was then used to the pixel intensity of the other channels (integrated density [IntDen]). Ciliary intensity of MC4R-GFP was then calculated as the IntDen of MC4R-GFP in the cilium, subtracting adjacent background (measured as IntDen of same defined area near the cilium). To calculate relative cilia enrichment, the IntDen (cilium) was divided by the IntDen (cell body), measured in the closest cell body (as defined by the presence of a Hoechst⁺ nucleus). Enrichment > 1, therefore, indicates higher localization of the receptor at the primary cilium compared with the cell body. Figure 2 details an in vitro experiment in which data are from 3 different replicates and are represented as a superplots, and n = 20–40 ciliated cells per condition were imaged and analyzed. Figure 4 indicates an in vivo experiment in which 60–90 cilia per mouse were quantified.

Z-stack images of MDCK monolayers (grown for 3 days on filters, and stained with anti-Arl13b antibody and Hoechst) were acquired on the Nikon A1R confocal microscope, with 0.28 μm pixel size and z-step. Analysis of the percentage of cells exhibiting an Arl13b-positive primary cilium was performed in FIJI, as follows: To count cilia, a maximum intensity projection of the Arl13b channel stack was taken, and rolling-ball background subtraction was applied with a radius of 100 pixels. The image was binarized using the Shanbhag thresholding algorithm, and particle analysis was applied to quantify the number of Arl13b puncta, analyzing particles with size ≥2 pixels. To count cells, a maximum intensity projection of the Hoechst channel stack was taken, the image was smoothed, and rolling-ball background subtraction was applied with a radius of 100 pixels. The image was binarized using the MinError(I) thresholding algorithm, and watershed separation was used to distinguish overlapping nuclei. Particle analysis was applied to quantify the number of nuclei, analyzing particles with size ≥150 pixels. The number of cilia was then divided by the number of nuclei to calculate the percent ciliation. 3–4 technical replicates (142 μm × 142 μm fields of view) were averaged from each biological replicate for each condition.