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. 2022 Apr 14;11:e74399. doi: 10.7554/eLife.74399

Figure 1. Centrioles in olfactory sensory neurons (OSNs) migrate tens of micrometers to the apical surface.

(A) Overview of the olfactory epithelium. Single-plane fluorescence image of a side view of the olfactory epithelium (left), corresponding to a schematic of cell types in the olfactory epithelium (right). Yellow: eGFP-centrin2; cyan: staining for acetylated tubulin, strongly marking olfactory cilia and, faintly, neuronal microtubules; magenta: staining for β-tubulin III, marking neuronal microtubules; white: DAPI, marking DNA. The apical surface is oriented at the top of this image and all subsequent side-view images. Dashed line: apical surface; double solid line: basal lamina. Boxes show the relative positions of critical stages of OSN differentiation: (i) the subapical compartment of the olfactory epithelium, defined as the space between the bottom of the sustentacular cell nuclei and the apical surface with olfactory cilia. (ii and inset) A group of centrioles migrating through the middle of the olfactory epithelium, below the sustentacular cell nuclei. (iii) A progenitor cell near the basal lamina. Scale bar = 10 μm. Inset scale bar = 2 μm. (B) Mature olfactory sensory neurons. Single-plane, en face fluorescence image of the apical surface of the olfactory epithelium. Green: eGFP-centrin2; magenta: dye-conjugated phalloidin, marking an enrichment of F-actin at the apical borders of sustentacular cells. Scale bar = 10 μm. (C) Schematic of (B) depicting a sustentacular cell wrapping around the dendrites of nearby OSNs. Green: OSN dendrite; gray: sustentacular cell cytoplasm; magenta: F-actin; wavy black lines: multiple OSN cilia. (D) Centriole migration in the olfactory epithelium. Single-plane fluorescence image of a side view of the subapical compartment of the olfactory epithelium. Green: eGFP-centrin2; magenta: dye-conjugated phalloidin; arrows: groups of migrating centrioles. Scale bar = 10 μm. (E) Mature olfactory sensory neurons, as imaged by expansion microscopy. Single-plane fluorescence image of the en face apical surface. White: staining for acetylated tubulin, marking centrioles, cilia, and, faintly, neuronal microtubules. Multiple cilia can be seen protruding from mature OSNs. A green arrow marks a dendrite with a group of migrating centrioles arriving at the apical tip. Scale bar = 2 μm. (F) Centriole migration, as imaged by expansion microscopy. Single-plane fluorescence image of a side view of the subapical compartment. White: staining for acetylated tubulin, marking centrioles, cilia, and, faintly, neuronal microtubules. Cilia can be seen at the apical surface, and green arrows mark groups of migrating centrioles. Scale bar = 2 μm.

Figure 1.

Figure 1—figure supplement 1. Centriole and cilium numbers in olfactory sensory neuron (OSN) progenitors and mature OSNs.

Figure 1—figure supplement 1.

(A) Example of Imaris segmentation of centrioles in progenitor cells. (i) 3D image of centrioles in a progenitor cell. Green: staining for acetylated tubulin. (ii) The same image after segmentation in Imaris. Segmented objects are color-coded with a LUT to encode z-depth. After automated counting, each image was visually inspected, and counts were corrected where objects and centrioles or cilia did not correspond. Scale bar = 2 µm. (B) Example of Imaris segmentation of centrioles and cilia in mature OSNs. (i) 3D image of a mature OSN dendritic knob. Green: staining for acetylated tubulin. (ii) The same image after segmentation in Imaris. Segmented objects are color-coded with a LUT to encode z-depth. After automated counting, each image was visually inspected, and counts were corrected where objects and centrioles or cilia did not correspond. Scale bar = 2 µm. (C) Plot of the number of centrioles per cell in progenitor cells and mature OSNs. Red dots: sample 1, 3-month-old female. Black dots: sample 2, 1-month-old male. Mean and standard error of the mean (SEM) are graphed. In progenitors: mean = 26.40 centrioles, SEM = 2.234 and standard deviation = 9.992. In mature OSNs: mean = 36.46 centrioles, SEM = 1.665, and standard deviation = 8.156. (D) Plot of centriole and cilium numbers in mature OSNs. Black dots: percentage of centrioles that have cilia. Purple squares: number of cilia in each cell. Each dot and square in the same column belong to the same dendritic knob. Measurements were arranged along the x-axis in order of increasing percentage of centrioles with cilia. Mean: 85% of centrioles at a dendritic knob nucleated cilia (SEM = 2.47; standard deviation = 12.10). Mean cilia number per dendritic knob: 30.54 cilia (SEM = 1.455; standard deviation = 7.126).