Table 1.
Antibody reagents.
Fig 1.
Schematic of experimental strategy to identify differential miRNA expression in olfactory epithelial cells.
(A) The olfactory epithelium, lining portions of the mouse nasal cavity, contains basal stem and progenitor cells, immature differentiating olfactory receptor neurons, mature neurons, sustentacular cells, and microvillar cells. (B) Following experimental lesion with methimazole, spared basal globose cells remain, and their expansion and differentiation (C) lead to rapid epithelial reconstitution. (D) Olfactory epithelial cells harvested 10 days following lesion are enriched with basal progenitors marked by surface expression of c-Kit. The c-Kit (+) basal cells were isolated by immunoselection, and total RNA was purified from the c-Kit (+) fraction or the c-Kit (-) fraction was analyzed by miRNA microarrays.
Table 2.
MiRNAs differentially downregulated in c-Kit (+ve) mouse globose cells post injury.
Table 3.
MiRNAs differentially upregulated in c-Kit (+ve) mouse globose cells post injury.
Fig 2.
Differential miRNA expession in cell populations of the olfactory epithelium.
Heatmap shows microarray results of top 28 miRNAs most downregulated (A) and top 10 miRNAs most upregulated (B) in c-Kit (+) versus c-Kit (-) fractions of mouse olfactory epithelium 10 days post olfactory lesion. N = 3 per group. Criteria used for selection is at least 4 fold change in transcript expression with p<0.05 based on t-test with Benjamini-Hochberg multiple testing correction. (C) QPCR validation shows miR-486 is 17 fold downregulated in c-Kit (+) versus (-) fractions of mouse olfactory epithelium 10 days post nasal injury. N = 3 per group. Data are Mean ± S.E.M. p = 0.001 based on t-test using delta Cts.
Fig 3.
Confirmation by in situ hybridization for miR-486 enrichment in the c-Kit (-) population.
MiR-486, identified as highly enriched in the c-Kit (-) cell fraction from regenerating olfactory epithelium, was chosen for further study to confirm the microarray findings. In situ hybridization was performed on normal adult mouse olfactory epithelium tissue sections; a miR-486 probe (A, B) reveals expression by cells throughout the neuronal layers (Neu), with little signal in the underlying lamina propria. At high magnification (B) an absence of signal in the c-Kit (+) basal cell layers (BCs) or in the apical sustentacular cell layers (Sus) is evident. (C) Hybridization using a scrambled control probe shows no signal. (D-K) Staining with cell type-specific markers was performed to define the OE cell populations. Antibody against c-Kit (D, E) labels only basal cells slightly above the basal lamina; antibody against OMP (F) labels mature neurons in the mid to upper portions of the OE; antibody against Tuj1 (G, H) labels the somata of immature neurons just above the basal cell layers, as well as dendrites extending apically; antibody against SOX2 (I) labels nuclei of a subset of basal cells near the basal lamina and also the sustentacular cell nuclei at the top of the epithelium; antibody against OAZ (J) localizes to nuclei of late GBCs and nascent neurons in deep layers of the OE; antibody against CK5 (K) labels HBCs along the basal lamina. (L) Cell layers and the markers used here are summarized schematically. Arrowheads or dashed line (in B) mark basal lamina; NEUm = mature neurons; NEUi = immature neurons; scale bar in B = 10 μm, in H = 20 μm.
Fig 4.
Functional effects of miR-486 in c-Kit-sorted progenitor basal cell culture preparations.
(A, B) Basal cells purified from OE of regenerating mice were seeded for short primary culture assay. Characterization of cultures at 24 hours confirms the presence of OE progenitors. Antibody against the neuron-specific transcription factor OAZ (magenta) labels the nuclei of neuron-committed GBCs and nascent or immature neurons; antibody against SOX2 (green) labels nuclei of cells scattered in basal cell islands, consistent with the growth of “upstream” undifferentiated GBCs. Culture composition is quantified in (B), n = 3. (C, D) Cultures were transfected after 24 hours with either miR486 or a scrambled control RNA. 48 hours post transfection, cells were fixed and stained for TUJ1 and DAPI and subsequently quantified. Although there was no significant change in proliferation by EdU incorporation (E), miR-486 treatment reduced the total amount of TUJ1 positive neurons by 30.05% ± 5.88 (p = 0.0014) normalized to scrambled control group (F); n = 8, mean ± S.E.M; student’s t-test. Bar = 20μm in A, 120μm in C and D.