DOI: 10.25378/janelia.29114576.v1
title: Near-isotropic, aligned volume electron microscopy (eFIB-SEM) of mouse choroid plexus (jrc_mus-choroid-plexus-3)

description: <b>Sample</b>: Wild-type, P14 mouse, strain: C57BL/6J<b>Sample Description</b>: The choroid plexus is a highly vascularized secretory neuroepithelium in brain ventricles, regulating the dynamic CSF production to support neuronal development and homeostasis. The monolayer of multiciliated cells in choroid plexus epithelia is essential for regulating CSF production. Aberrant ciliogenesis or defective intraflagellar transport in choroid plexus epithelia can disrupt CSF homeostasis, leading to pathology of hydrocephalus or microcephalus. A previous study characterize mature choroid plexus multicilia are nodal-like “9+0”non-motile multicilia that contains features of both primary and motile cilia. However, the traditional TEM has z-limitation in 3D structure and may underestimate the ultrastructure of choroid plexus cilia. Thus, we used Focused Ion Beam Scanning Electron Microscopy (FIB-SEM) to further analyses analyzed 17 mature P14 Choroid plexus epithelia cells (CPECs). We obtained 286 imaged basal bodies and modeled the CPEC ciliary and basal body ultrastructure in 3D. P14 CPECs exhibited heterogeneity in the numbers of cilia, with an average of ~18 cilia and ~18 basal bodies per cell Strikingly, choroid plexus also exhibited a heterogeneous basal body assembly where varying number of SDAs are asymmetrically arranged with no ciliary attachment, yet usually with one dominant SDA larger in size. Interestingly, such asymmetric-arrange SDA displayed unique cellular distribution, where basal bodies with more SDAs (4SDA or 5SDA) were often localized in the center of the basal body network, while those with fewer SDAs (2SDAs or 3SDAs) were frequently in the periphery. Collectively, our FIB-SEM study on mature choroid plexus cilia comprehensively and accurately depict their distinct ultrastructure, highlighting their unique basal body network as a structural basis for their biological functions.<b>Protocol</b>: Choroid plexus samples were first fixed in 2.5% formaldehyde and 2.5% glutaraldehyde in 0.1M Sodium Cacodylate Buffer, PH=7.4 (Emsdiasum, Cat# 15949), and then stained with a modified osmium-thiocarbohydrazide-osmium (OTO) method in combination with microwave assisted processing, followed by HPF-FS as previously described 10. Briefly, samples were subjected to HPF-FS, and freeze-substituted with 4% osmium tetroxide, 0.1% uranyl acetate, and 5% ddH2O in acetone; then thin embedded and polymerized in Durcupan resin.<b>Contributions</b>: Sample provided by Suifang Mao, Rui Song, and Shibo Jin (UC Berkeley), prepared for imaging by Suifang Mao and Rui Song (UC Berkeley), with imaging and post-processing by C. Shan Xu (HHMI/Janelia), Srigokul Upadhyayula (UC Berkeley), and Harald Hess (HHMI/Janelia).<b>Acquisition ID</b>: jrc_mus-choroid-plexus-3<b>Voxel size (nm)</b>: 8 x 8 x 8 (x, y, z)<b>Data dimensions (µm)</b>:<b> </b>60 x 60 x 60.48 (x, y, z)<b>Imaging start date</b>: 2018-06-05<b>Imaging duration (days)</b>: 18<b>Landing energy (eV)</b>: 1500<b>Imaging current (nA)</b>: 3<b>Scanning speed (MHz)</b>: 1.250<b>Dataset URL</b>: s3://janelia-cosem-datasets/jrc_mus-choroid-plexus-3/jrc_mus-choroid-plexus-3.zarr/recon-1/em/<b>Visualization Website</b>: https://openorganelle.janelia.org/datasets/jrc_mus-choroid-plexus-3<b>Publication</b>:<b> </b>Mao et al., 2025; Xu et al., 2017<br>