Fig 1.
Experimental design and data analysis.
(A) Schematic Representation: (Top Left) Mice were perfused and harvested at two-hour intervals over a 24-h period. The harvested mice were then sectioned coronally. (Bottom Left) Coronal brain sections at different brain levels. (Top Middle) The ciliary base was defined as the origin of the coordinate system, and the tip of the cilia was labeled. Cilia length was measured from the base to the tips, and the circular angle was determined. (Bottom Middle) The brain section of the mice was divided into left and right hemispheres. For cilia found in the right hemisphere, a counter-clockwise coordinate system was used to determine the circular angle (Right). For cilia found in the left hemisphere, a clockwise coordinate system was used (Left). (Top Right) An example of the angle measurement from cilia found in the right hemisphere. (B) Representative images of immunofluorescence staining showing cilia labeled with the ADCY3 antibody (red) and the basal body (cilia base) labeled with the centrin 1 antibody (green). (C) Brain Regions analyzed for cilia length and angle. (D) The number of cilia analyzed in 22 different brain regions of the mice; underlying data are available on the Zenodo database (https://doi.org/10.5281/zenodo.15151271). (E) Cilia density in 22 brain regions, expressed as the percentage of ciliated cells relative to the total number of cells in each region, underlying data are available in S1 Data. Parts of Fig 1A are created with BioRender.com.
Fig 2.
Cilia length analysis in different brain regions.
(A) Histogram showing the means±SE of cilia length in the 22 brain regions based on the average length measured in each brain section. The x-axis represents cilia length in micrometers and the y-axis represents the percentage frequency of occurrence. (B and C) Histograms showing the frequency distribution of cilia length in (B) the entire brain and (C) the 22 individual brain regions, using the section-based average method. For each brain section, the lengths of all cilia were averaged to obtain a single value, which was then used to calculate the overall mean cilia length for each region. The mean cilia length was derived from the average lengths of both left and right hemisphere sections (3–4 sections per region). The x-axis represents cilia length in micrometers, and the y-axis shows the percentage frequency of occurrence; underlying data for Fig 2 are available in S2 Data.
Fig 3.
Cilia circular angle analysis, based on individual cilia measurements.
Rose plot displaying the frequency distribution of cilia circular angles in individual brain regions from all individual measured cilia at 30° bins, Underlying data are available on the Zenodo database (https://doi.org/10.5281/zenodo.15151271).
Fig 4.
Cilia circular angle analysis based on section-averaged circular means.
(A) Radial plot showing the frequency distribution of cilia circular angles across the entire brain and individual brain regions. The analysis is based on section-averaged circular means, where the circular mean of all cilia angles within each section was first calculated and treated as a single value. Underlying data are available in S2 Data. (B) Radial plots displaying the mean cilia circular angles for the 22 brain regions, grouped into major categories: hypothalamus, hippocampus, cortices, and striatum. The average angle for each region was determined by calculating the circular angle means for each brain section. Underlying data are available in S2 Data.
Fig 5.
Fluctuation of cilia length and cilia circular angle across 24-hours’ time.
(A and B) Diurnal fluctuations of cilia length (A) and angle (B): The plots show the means ± SE of cilia length and angle over the course of 24-h day in different brain regions. The brain regions are grouped into major categories including, the Hypothalamus, Hippocampus, Cortices, and Striatum. The average length and angle are determined based on the measurements’ means in each brain section; Zeitgeber time (ZT). One-way ANOVA test was used to compare the means of lengths and angles at different time points. P values were calculated using the False Discovery Rate (FDR) correction, employing the Two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli for multiple comparisons. (C) P values of one-way ANOVA test, used to assess the variability in cilia length and angle at different time points (in a,b), P < 0.05: significant changes across the different time points over 24-h period. Underlying data for Fig 5 are available in S2 Data.
Fig 6.
Circadian rhythms of cilia length.
BioCycle analysis was used to examine the presence of rhythmic fluctuations in cilia length and revealed significant circadian patterns of cilia length variations across a 24-h period in five brain regions tested: ARC, DMH, VMH, NAc, and SSC, with P < 0.05. Underlying data are available in S3 Data.
Fig 7.
Correlation of cilia length and circular angle among various brain regions and relationship between cilia length/angle and network connectivity.
(A) Heatmap showing the correlation of cilia length (based on the mean of the section means) among different brain regions. The correlation coefficient ranges from 1 (indicating the most positive correlation, red) to −1 (indicating the most negative correlation, blue). Positive correlation implies that the cilia lengths in the two regions are on the longer and shorter side simultaneously. Negative correlation indicates that the two regions have opposite cilia length sizes; underlying data are available in S2 Data. (B) Heatmap displaying the correlation of cilia angle (based on the mean of the section circular means) among different brain regions. The correlation coefficient ranges from 1 (indicating the most positive correlation, purple) to −1 (indicating the most negative correlation, green); underlying data are available in S2 Data. (C) Anatomical network of the brain regions in our study. The network is embedded in 2D space using a spring embedding, in which highly connected nodes are pulled together as by a spring. Node size represents betweenness, a measurement of centrality based on the number of shortest paths between nodes that a particular node shows up on. Nodes are colored according to their community assignment, as determined by the Leiden algorithm. (D) Correlogram indicating the strength of input-output connections between all pairs of regions in the dataset. The color bar indicates the log-normalized projection density between any two regions. Regions are grouped according to their community membership. (E) Bar graph comparing projection strength between regions significantly correlated with cilia length vs. those not correlated. Projection strength equals the log normalization of normalized (by injection volume) projection density. (F) Fraction of region pairs significantly correlated by cilia length among all regions pairs between two communities. For example, a fraction of 0.111 means that 11.1% of all region pairs between the red and green communities are correlated by cilia length. (G) Bar graph comparing projection strength between regions significantly correlated with cilia angle versus those not correlated. (H) Fraction of region pairs significantly correlated by cilia angle among all regions pairs between two communities. (I) Cilia length correlation network. The spring embedding puts regions more highly correlated closer together. Betweenness and community membership are shown as node size and color, respectively. (J) Cilia length correlogram shows the groupings of different regions by community, as well as their correlation patterns. (K) Cilia angle correlation network. Betweenness and community membership are shown as node size and color, respectively. (L) Cilia angle correlogram shows the groupings of different regions by community, as well as their correlation patterns. Underlying data of Fig 7C-L are available in S2 Data, and from the publicly available Allen Mouse Brain Connectivity Atlas Data.