Fig 1.
Location of rosette structures and free centrioles in adult and embryonic mouse olfactory epithelium.
(A) Schematic of the olfactory epithelium. The inset shows an OSN dendrite from adult mouse imaged by TEM with pseudocolored cilia and centrioles. Inset scale bar = 1 μm. (B) TEM image of wild-type adult mouse olfactory epithelium. Double solid line marks the basal lamina. Box shows the approximate location of the inset in the panel to the right. The panel on the right shows an inset of a centriole rosette in cross section, near the basal lamina. Scale bar = 10 μm. Inset scale bar = 0.5 μm. (C) Fluorescence image of embryonic olfactory epithelium at E12.5 in mice expressing eGFP-centrin2 to mark centrioles, as well as Arl13b-mCherry to mark cilia. The maximum projection inset shows a deconvolved image of 2 rosette-like centriole clusters from a cell positive for β tubulin III, near the basal lamina. Dashed line marks the apical surface of the olfactory epithelium. Double solid line marks the basal lamina. Box shows the location and orientation of the inset. Scale bar = 20 μm. Inset scale bar = 1 μm. (D) TEM images of wild-type adult mouse olfactory epithelium in serial sections. The images show 2 centriole rosettes, R1 and R2, and free centrioles, C1-4. Section sequence is indicated in the bottom right of each panel. The bottom panel summarizes the locations of centrioles in all 4 panels, where new centrioles are shown in purple and mother centrioles are shown in gray. Scale bar = 1 μm. See S1 Fig for additional details. Arl13b-mCherry, ADP-ribosylation-factor-like GTPase 13b conjugated to mCherry; eGFP, enhanced green fluorescent protein; nuc, nucleus; OSN, olfactory sensory neuron; TEM, transmission electron microscopy.
Fig 2.
Division of cells with amplified centrioles.
(A) Maximum projection image of immunofluorescence in olfactory epithelium cryosections from an adult mouse expressing eGFP-centrin2. Dashed line marks the apical surface of the olfactory epithelium. Double solid line marks the basal lamina. Box marks the location of the inset shown in A’. Scale bar = 5 μm. Inset (A’) shows a deconvolved maximum projection of centrioles shown in panel A. eGFP-centrin2 and γ tubulin mark 2 clusters of centrioles. Phospho-H3 marks a cell in an early phase of mitosis. DAPI shows DNA condensed near the basal lamina and is excluded from the merge. Inset scale bar = 2 μm. (B) Immunofluorescence in cryosections of olfactory epithelium from a wild-type adult mouse. DAPI shows DNA condensed and aligned in metaphase with strong phospho-H3 colocalization. Centrin marks the distal ends of centrioles. In this single optical section, centriole clusters are shown at the spindle poles, marked by acetylated tubulin. DNA is excluded from the merge. Scale bar = 2 μm. (C) Single optical section image of immunofluorescence in olfactory epithelium from a wild-type adult mouse. Diffuse phospho-H3 marks a cell in anaphase. White arrows denote clusters of centrioles on opposite sides of the dividing cell. Scale bar = 2 μm. (D) Maximum projection of a deconvolved fluorescence image in olfactory epithelium from a mouse at embryonic stage E12.5. DAPI shows DNA condensed and aligned in metaphase. One spindle pole is shown with a cluster of centrioles marked by eGFP-centrin2. Scale bar = 2 μm. (E) Fluorescence image of a mouse at embryonic stage E12.5. DAPI shows DNA decondensing to form 2 daughter cell nuclei. White lines show cell boundaries, including a cleavage furrow, as approximated by cytoplasmic eGFP-centrin2 resulting from overexpression. Boxes mark the location of inset images. Scale bar = 5 μm. The insets (E’ and E”) show deconvolved maximum projection images of centriole clusters in the top and bottom boxes, respectively. Centrioles are marked by eGFP-centrin2. Inset scale bars = 2 μm. See S2 Fig for additional details. Arl13b-mCherry, ADP-ribosylation-factor-like GTPase 13b; eGFP, enhanced green fluorescent protein; phospho-H3, phosphorylated histone 3.
Fig 3.
Plk4 and Stil RNA levels and centriole number in early immediate neuronal precursors in the olfactory epithelium.
(A-C) Secondary analysis of an existing single-cell RNA sequencing data set from Fletcher and colleagues compares RNA levels for specific genes across cell types in the olfactory epithelium [21]. The vertical axis shows average log2(normalized RNA counts). Each dot represents one cell. The horizontal axis shows cell groups in the pseudotime lineage order determined by Fletcher and colleagues and is summarized at the top of panel A. (A) RNA levels for Rrm2, a gene specific to DNA synthesis in S phase. (B) RNA levels for genes that drive centriole formation Plk4 and Stil. (C) RNA levels for NeuroD1, a transcription factor marking early immediate neuronal precursor cells. Center lines = mean. Error bars = standard deviation. See S2 Data for values. (D-E) Fluorescence images of olfactory epithelium from adult mice expressing eGFP-centrin2 and Arl13b-mCherry. Cryosections were stained with antibodies against NeuroD1 and γ tubulin and with DAPI to mark DNA. (D) A NeuroD1-positive cell with two centrioles. (E) A NeuroD1-positive cell with greater than two centrioles. DNA is excluded from the merge. Scale bars = 5 μm. (F) Comparison of centriole counts in different cell types from olfactory epithelium of adult mice expressing eGFP-centrin2 and Arl13b-mCherry. Centrioles in NeuroD1-positive cells were counted in dissociated olfactory epithelia (N = 2 mice, n = 40 cells). Centrioles in OSNs were counted by en face imaging of the apical surface of septum olfactory epithelia (N = 6 mice, n = 90 cells). Note the small population of NeuroD1-positive cells with unamplified centrioles. See S3 Data for centriole counts. See S3 Fig for additional details. Arl13b-mCherry, ADP-ribosylation-factor-like GTPase 13b; eGFP, enhanced green fluorescent protein; NeuroD1, neuronal differentiation 1; OSN, olfactory sensory neuron; Plk4, polo-like kinase 4; Rrm2, ribonucleotide reductase molecule 2; Stil, SCL/Tal1 interrupting locus gene.
Fig 4.
Summary of centriole amplification in the olfactory epithelium.
OSN, olfactory sensory neuron; Plk4, polo-like kinase 4.
Table 1.
Mice and cells used.
Table 2.
Primary antibodies used.