Subcellular analysis of pigeon hair cells implicates vesicular trafficking in cuticulosome formation and maintenance
- Simon Nimpf
- Erich Pascal Malkemper
- Mattias Lauwers
- Lyubov Ushakova
- Gregory Nordmann
- Andrea Wenninger-Weinzierl
- Thomas R Burkard
- Sonja Jacob
- Thomas Heuser
- Guenter P Resch
- David A Keays
- Vienna Biocenter, Austria
- Vienna BioCenter Core Facilities GmbH, Austria
- Nexperion e.U., Austria
Figures
Figure 1 with 6 supplements
Development and cellular localization of cuticulosomes.
(a) Schematic representation showing the localization of iron-rich organelles in hair cells viewed from the side and the top. The stereocilia (sc), the nucleus (nc) and the cuticular plate (cp) are …
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Figure 1—source data 1
Quantitation of Prussian blue positive hair cells in the basilar papilla and lagena macula.
This table shows the estimated numbers of total hair cells, total PB +ve hair cells and the percentage of PB +ve hair cells in the basilar papilla and lagenar macula of all pigeons used for the analysis shown in Figure 1c and d and Figure 1—figure supplements 2–6 ‘R’ and”L’ stand for ‘right’ or ‘left’ cochlear duct.
- https://doi.org/10.7554/eLife.29959.009
Figure 1—figure supplement 1
Developmental time line of iron-rich organelles.
(a–j) Images of histological sections from the basilar papilla and lagenar macula of 1 day (a and b), 8 days (c and d), 16 days (e and f), 30 days (g and h) and 1 year old pigeons (i and j) stained …
Figure 1—figure supplement 2
Quantitation and distribution of iron-rich organelles in the cochlear duct of 1 day old pigeons.
Graphs show the percentage of PB +ve hair cells along the length of the basilar papilla (shown in red) and the lagena (shown in blue) in 40 µm increments from four individual birds.
Figure 1—figure supplement 3
Quantitation and distribution of iron-rich organelles in the cochlear duct of 8 day old pigeons.
Graphs show the percentage of PB +ve hair cells along the length of the basilar papilla (shown in red) and the lagena (shown in blue) in 40 µm increments from three individual birds.
Figure 1—figure supplement 4
Quantitation and distribution of iron-rich organelles in the cochlear duct of 16 day old pigeons.
Graphs show the percentage of PB +ve hair cells along the length of the basilar papilla (shown in red) and the lagena (shown in blue) in 40 µm increments from six individual birds.
Figure 1—figure supplement 5
Quantitation and distribution of iron-rich organelles in the cochlear duct of 30 day old pigeons.
Graphs show the percentage of PB +ve hair cells along the length of the basilar papilla (shown in red) and the lagena (shown in blue) in 40 µm increments from five individual birds.
Figure 1—figure supplement 6
Quantitation and distribution of iron-rich organelles in the cochlear duct of 1 year old pigeons.
Graphs show the percentage of PB +ve hair cells along the length of the basilar papilla (shown in red) and the lagena (shown in blue) in 40 µm increments from three individual birds.
Figure 2 with 1 supplement
Subcellular architecture of iron-rich organelles during development.
(a–h) Electron micrographs showing cuticulosomes in the cuticular plate (cp) apically located beneath the stereocilia (sc) in hair cells from the basilar papilla of 1 day (a and f), 8 day (b and g), …
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Figure 2—source data 1
Properties of cuticulosomes at different ages.
This table shows the percent of cuticulosomes that are surrounded by membranes, associated with vesicles, have a paracrystalline organisation, and those that are incomplete at 1 day, 8 days, 16 days, 30 days and 1 year. Data for 1 year was taken from (Lauwers et al., 2013).
- https://doi.org/10.7554/eLife.29959.012
Figure 2—figure supplement 1
Density analysis of the cuticular plate.
(a) Electron micrograph of a cuticulosome of an 8 day old pigeon. Lines were drawn (200 nm in length) radially from the edge of cuticulosomes at 45° intervals. The mean density along these lines was …
Figure 3 with 4 supplements
Tomograms and three-dimensional reconstructions of cuticulosomes in 8 days old pigeons.
(a, d, g, j) Images of 9 nm thick sections computational derived from tomograms of cuticulosomes from the basilar papillar of pigeons aged 8 days (n = 3 birds, n = 4 cells). (b, e, h, k) …
Figure 3—video 1
Tomographic reconstruction of a developing cuticulosome.
This movie shows a tomographic model of a developing cuticulosome in a 8 day old pigeon. The model was generated from a dual axis tilt series with a tilt range from −60° to +60° with one degree …
Figure 3—video 2
Tomographic reconstruction of a developing cuticulosome.
This movie shows a tomographic model of a developing cuticulosome in a 8 day old pigeon. The model was generated from a dual axis tilt series with a tilt range from −60° to +60° with one degree …
Figure 3—video 3
Tomographic reconstruction of a developing cuticulosome.
This movie shows a tomographic model of a developing cuticulosome in a 8 day old pigeon. The model was generated from a dual axis tilt series with a tilt range from −60° to +60° with one degree …
Figure 3—video 4
Tomographic reconstruction of a developing cuticulosome.
This movie shows a tomographic model of a developing cuticulosome in a 8 day old pigeon. The model was generated from a dual axis tilt series with a tilt range from −60° to +60° with one degree …
Figure 4 with 2 supplements
Heavy chain ferritin is a component of the cuticulosome.
(a–c) Transmitted light images of sections of the basilar papilla of 1 day, 8 days and 1 year old pigeons. Cuticulosomes are highlighted with black arrows. (d–f) Confocal images of the same …
Figure 4—figure supplement 1
Ferritin expression during development.
Quantitative real-time PCR results for Ferritin heavy chain from isolated hair cell layers of pigeons aged 1 day, 8 days, 16 days, 30 days and 1 year (n = 3 birds per timepoint). Expression levels …
Figure 4—figure supplement 2
Quantitation of Turnbull blue positive hair cells.
(a and c) Images of histological sections from the basilar papilla (a) and lagenar macula (c) of a 16 days old pigeon stained with Turnbull blue (TB), intensified with DAB and counterstained with …
Figure 5 with 1 supplement
Transcriptomic analysis of hair cells with or without cuticulosomes.
(a) Diagram showing the methodology employed for transcriptomic analysis. Following the dissection of the cochlear duct and removal of the lagena, the basilar papilla and surrounding tissue were …
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Figure 5—source data 1
Transcripts Identified by RNA Sequencing.
List of all genes that were identified in the RNA sequencing experiment (mean FPKM >1), showing the log2-fold change between cuticulosome positive and negative hair cells, adjusted P-values, and expression levels (FPKM) for all individual samples.
- https://doi.org/10.7554/eLife.29959.023
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Figure 5—source data 2
Significantly differentially expressed genes between hair cells with and without iron-rich organelles.
This table shows a list of adjusted p-values, log2-fold changes and general information about the genes that were significantly, differentially expressed between hair cells with and without cuticulosomesomes.
- https://doi.org/10.7554/eLife.29959.024
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Figure 5—source data 3
GO enrichment analysis for ‘cellular component’.
This table shows all GO terms (cellular component) that were significantly enriched in genes that were upregulated in cuticulosome positive hair cells (>3 fold).
- https://doi.org/10.7554/eLife.29959.025
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Figure 5—source data 4
GO enrichment analysis for ‘molecular function’.
This table shows all GO terms (molecular function) that were significantly enriched in genes that were upregulated in cuticulosome positive hair cells (>3 fold).
- https://doi.org/10.7554/eLife.29959.026
Figure 5—figure supplement 1
Analysis of RNA sequencing results.
(a and b) Graphs show the 20 highest expressed genes in cuticulosome positive and negative hair cells (mean FPKM, n = 9 per cell type). (c) Graph showing the expression levels (mean FPKM, all …
Figure 6
Model of cuticulosome development.
(a) Cuticulosomes start to form shortly after hatching at the lateral apical edge of the cuticular plate, opposite the kinocilium. They initially resemble electron dense accumulations of amorphous …
Additional files
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Transparent reporting form
- https://doi.org/10.7554/eLife.29959.028
