Single-cell proteomics reveals changes in expression during hair-cell development
- Pacific Northwest National Laboratory, United States
- Stanford University, United States
- Amgen Inc, United States
- Oregon Health & Science University, United States
- Kyung Hee University, Republic of Korea
- Brigham Young University, United States
Figures
Figure 1 with 2 supplements
Mass spectrometry of single cells and small cell pools from E15 chick utricle.
(A) Experimental design. The E15 chick utricle’s sensory epithelium consists of sensory hair cells and supporting cells, which are also progenitor cells. FM1-43 labels hair cells more strongly than …
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Figure 1—source data 1
MaxQuant analysis of single cell proteomics data.
Excel file containing data from Experiments 1 and 2 analyzed together by MaxQuant and Andromeda.
- https://doi.org/10.7554/eLife.50777.005
Figure 1—figure supplement 1
Characterization of isolated utricle cells.
(A-C) FACS gating protocol. (A) Debris was removed based on side-scatter area (SSC-A) and forward-scatter area (FSC-A). Blue box shows cells collected. (B) Doublets were discarded based on forward …
Figure 1—figure supplement 2
Peptide coverage of TMSB4X in mass spectrometry experiments.
Characterization of isolated utricle cells. (A–C) FACS gating protocol. (A) Debris was removed based on side-scatter area (SSC-A) and forward-scatter area (FSC-A). Blue box shows cells collected. (B)…
Figure 2 with 3 supplements
Abundant proteins in small pools of isolated E15 chick utricle cells.
(A) Heat map showing top 60 proteins or protein groups in samples of 20 cells, sorted by the average of the 20 cell FM1-43high samples. FM1-43low and FM1-43high samples from Experiment 1 (Exp1) and …
Figure 2—figure supplement 1
Expression levels for all proteins in samples from Experiments 1 and 2 that contain pools of three cells, five cells, or 20 cells.
Each sample (indicated by letters) under the callout for cell numbers contains that number of cells (i.e., on the left, samples a-c under labels indicating ‘Experiment 1’ and ‘FM1-43 low’ each …
Figure 2—figure supplement 2
Expression levels for all proteins in samples from Experiments 1 and 2 from single-cell samples.
Each sample is indicated by letters. ‘Avg’ indicates the average of the 20 cell samples for that condition and experiment. Log10 riBAQ color scale is indicated at bottom. Heat map is sorted by the …
Figure 2—figure supplement 3
Expression levels for all proteins in single-cell samples in Experiments 1 and 2.
Each sample is indicated by letters. ‘Avg’ indicates the average of the 20 cell samples for that condition and experiment. Heat map, which shows all identified proteins or protein groups in single …
Figure 3 with 6 supplements
Immunolocalization of proteins enriched in hair cells or supporting cells of E15 chick utricle.
Confocal z-stacks of vibratome cross-sections of the whole utricle were imaged with the tiling and stitching function in Zeiss ZEN. Confocal z-stacks for magnified extrastriolar and striolar regions …
Figure 3—figure supplement 1
Immunolocalization of AGR3 in E15 chick utricle.
The sensory epithelium and the stroma are indicated in the transmitted-light section. Nuclei were stained with DAPI and F-actin with phalloidin; OTOF and AGR3 were stained with specific antibodies. …
Figure 3—figure supplement 2
Immunolocalization of CRABP1 in E15 chick utricle.
The sensory epithelium and the stroma are indicated in the transmitted-light section. Nuclei were stained with DAPI and F-actin with phalloidin; MYO7A and CRABP1 were stained with specific …
Figure 3—figure supplement 3
TMSB4X localization.
The sensory epithelium and the stroma are indicated in the transmitted-light section. Nuclei were stained with DAPI and F-actin with phalloidin; OTOF and TMSB4X were stained with specific …
Figure 3—figure supplement 4
G-actin localization with JLA20 antibody.
The sensory epithelium and the stroma are indicated in the transmitted-light section. Nuclei were stained with DAPI and F-actin with phalloidin; MYO7A and G-actin were stained with specific …
Figure 3—figure supplement 5
OCM localization with anti-PV3 antibody.
The sensory epithelium and the stroma are indicated in the transmitted-light section. Nuclei were stained with DAPI and F-actin with phalloidin; OTOF and OCM were stained with specific antibodies. …
Figure 3—figure supplement 6
Identification of striola and extrastriola regions.
The sensory epithelium and the stroma are indicated in the transmitted-light section. Nuclei were stained with DAPI and F-actin with phalloidin; SOX2, MYO7A and tubulin beta-3 (TUJ1) were stained …
Figure 4
Pseudotemporal ordering of single utricle cells based on proteomics measurements.
(A) Relationship between variance and mean expression, distinguishes proteins or protein groups with low variance (blue) or high variance (salmon). (B) First three components of CellTrails' spectral …
Figure 5
Developmental trajectory identified from single utricle cell RNA-seq measurements.
(A) CellTrails identifies nine distinct states (cellular subgroups). Shown are 328 points representing single utricle cells projected into two-dimensional space using CellTrails t-distributed …
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Figure 5—source data 1
CellTrails analysis of single-cell RNA-seq data.
Excel file including: Tab S1A: Single-cell RNA-seq measurements. Count matrix after QC and Normalization (ScNorm) of 186 genes and 328 chicken utricle single cells. Rows display single cells and columns display genes. Non-detected signals were set to 0. Tab S1B: tSNE plots and CellTrails maps. Listed are tSNE plots and CellTrails maps for each gene. The first column lists the gene symbol names, the second column shows the tSNE plots, the third column shows the CellTrails maps with the raw expression values, the fourth column shows the CellTrails maps with smoothed expression values, and the fifth column shows the CellTrails maps with the topographical expression surface. Tab S1C: Inferred expression dynamics. Shown is individual pseudotime gene expression along the extrastriolar (TrES, TrES*) and striolar (TrS) trajectory.
- https://doi.org/10.7554/eLife.50777.020
Tables
Table 1
Summary statistics with confidence intervals for Figure 2C.
https://doi.org/10.7554/eLife.50777.017| 95% Confidence interval | |||||||
|---|---|---|---|---|---|---|---|
| Comparison | Estimate | Std. error | df | t-value | Lower bound | Upper bound | p-value |
| ACTG1 FM-high – ACTG1 FM-low | −0.017 | 0.009 | 22.3 | −1.830 | −0.036 | 0.002 | 0.081 |
| ACTG1 FM-high – TMSB4X FM-high | 0.036 | 0.008 | 12.0 | 4.607 | 0.019 | 0.054 | 0.001 |
| ACTG1 FM-low - TMSB4X FM-low | 0.004 | 0.008 | 12.0 | 0.451 | −0.014 | 0.021 | 0.66 |
| TMSB4X FM-low - TMSB4X FM-low | −0.050 | 0.009 | 22.3 | −5.356 | −0.069 | −0.031 | <0.001 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Biological sample (Gallus gallus) | Embryonic day 15 utricle | eggs from Texas A and M University Poultry Science Department | n/a | Freshly isolated from Gallus gallus |
| Chemical compound, drug | FM1-43FX | Thermo Fisher | Cat# F35355 | 10 µM |
| Chemical compound, drug | SYTOX Red Dead Cell Stain | Thermo Fisher | Cat# S34859 | 1:1000 (final 5 nM) |
| Chemical compound, drug | thermolysin from geobacillus stearothermophilus | Sigma-Aldrich | Cat# T7902 | 0.5 mg/ml |
| Chemical compound, drug | Accutase | Innovative Cell Technologies | Cat# AT104 | full strength |
| Chemical compound, drug | n-dodecyl β-D-maltoside | Sigma-Aldrich | Cat# D4641 | 0.1% (w/v) |
| Chemical compound, drug | trypsin | Promega | Cat# V5280 | 10 ng/µl |
| Chemical compound, drug | Lys-C | Promega | Cat# V1671 | 10 ng/µl |
| Chemical compound, drug | DAPI | Thermo Fisher | Cat# D1306 | 1 µg/ml |
| Chemical compound, drug | Alexa Fluor 488-conjugated phalloidin | Thermo Fisher | Cat# A12379 | 1:1000 |
| Antibody | rabbit polyclonal anti-TMSB4X | Proteintech | Cat# 19850–1-AP, RRID: AB_10642437 | 1:250 |
| Antibody | rabbit polyclonal anti-AGR3 | Proteintech | Cat# 11967–1-AP, RRID: n/a | 1:250 |
| Antibody | rabbit polyclonal anti-CRABP1 | Proteintech | Cat# 12588–1-AP, RRID: AB_2292271 | 1:250 |
| Antibody | mouse monoclonal anti-G-actin | Developmental Studies Hybridoma Bank | Cat# JLA20, RRID: AB_528068 | 1:250 |
| Antibody | mouse monoclonal anti-otoferlin | Developmental Studies Hybridoma Bank | Cat# HCS-1, RRID: AB_10804296 | 1:250 |
| Antibody | goat polyclonal anti-SOX2 | Santa Cruz Biotechnology | Cat# sc-17320, RRID: AB_2286684 | 1:100 |
| Antibody | mouse monoclonal anti-tubulin beta-3 | BioLegend | Cat# TUJ1, RRID: AB_2313773 | 1:250 |
| Antibody | rabbit polyclonal anti-parvalbumin-3/oncomodulin | Heller laboratory | n/a | 1:1000 |
| Antibody | rabbit polyclonal anti-MYO7A | Proteus Biosciences | Cat# 25–6790, RRID: AB_2314838 | 1:1000 |
| Antibody | Alexa Fluor 546 donkey anti-rabbit polyclonal | Thermo Fisher | Cat# A10040, RRID: AB_2534016 | 1:250 |
| Antibody | Alexa Fluor 647 donkey anti-mouse polyclonal | Thermo Fisher | Cat# A31571, RRID: AB_162542 | 1:100 |
| Antibody | Alexa Fluor 488 donkey anti-goat polyclonal | Thermo Fisher | Cat# A11055, RRID: AB_2534102 | 1:250 |
| Commercial assay or kit | SMARTscribe | Clontech | Cat# 639538 | |
| Commercial assay or kit | Hifi HotStart ReadyMix (2X) | Kapa Biosystems | Cat# KK2602 | |
| Software, algorithm | MaxQuant | Cox lab, Max Planck Institute of Biochemistry | https://www.maxquant.org | |
| Software, algorithm | FIJI (ImageJ) | n/a | http://fiji.sc/ | |
| Software, algorithm | R | The R Project for Statistical Computing | https://www.r-project.org/ | |
| Software, algorithm | limma R package | Bioconductor | DOI: 10.18129/B9.bioc.limma | |
| Software, algorithm | sva R package | Bioconductor | DOI: 10.18129/B9.bioc.sva | |
| Software, algorithm | CellTrails R package | Bioconductor | DOI: 10.18129/B9.bioc.CellTrails | |
| Other | Medium 199 | Thermo Fisher | Cat# 12350039 | |
| Other | SPHERO Drop Delay Calibration Particles | Spherotech | Cat# DDCP-70–2 | |
| Other | nanoPOTS chips | Custom built | n/a |
Table 2
Antibodies used.
https://doi.org/10.7554/eLife.50777.021| Antibody | Dilution | Supplier | Catalog # | Validation |
|---|---|---|---|---|
| rabbit anti-TMSB4X | 1:250 | Proteintech | 19850–1-AP | (Zhou et al., 2013; Li et al., 2018) |
| rabbit anti-AGR3 1:250 | 1:250 | Proteintech | 11967–1-AP | From manufacturer’s website: correct-sized band by protein immunoblot in SKOV-3 cells, MCF-7 cells (chicken protein 88% identical to mouse) |
| rabbit anti-CRABP1 1:250 | 1:250 | Proteintech | 12588–1-AP | From manufacturer’s website: correct-sized band by protein immunoblot in human spleen tissue, transfected HEK-293 cells (chicken protein 95% identical to human) |
| mouse anti-G-actin 1:250 | 1:250 | Developmental Studies Hybridoma Bank | JLA20 | (Lin, 1981) |
| mouse anti-otoferlin 1:250 | 1:250 | Developmental Studies Hybridoma Bank | HCS-1 | (Goodyear et al., 2010) (chicken protein 73% identical to mouse) |
| goat anti-SOX2 1:100 | 1:100 | Santa Cruz Biotechnology | sc-17320 | From manufacturer’s website: ‘recommended for detection of Sox-2 of mouse, rat, human and avian origin by WB, IP, IF, IHC(P) and ELISA’ |
| mouse anti-tubulin beta-3 TUJ1. 1:250 | 1:250 | BioLegend | 801202 | (Lee et al., 1990) |
| rabbit anti-parvalbumin3/oncomodulin | 1:1000 | Heller lab | 16910 | (Heller et al., 2002) |
| rabbit anti-MYO7A | 1:1000 | Proteus Biosciences | 25–6790 | (Morgan et al., 2016) |
Table 3
Genes included in scRNA-seq CellTrails analysis.
https://doi.org/10.7554/eLife.50777.022| ABCA5 | (WHRN) | (NSG2) | PLSCR5 | TMSB4X |
|---|---|---|---|---|
| ACO1 | DFNB59 | LOC423919 | PNPT1 | TNNC2 |
| ACTB | (PJVK) | (SHTN1) | PODXL2 | TOLLIP |
| ACTG1 | DIAPH1 | LOC772075 | POU4F3 | TPM1 |
| ACTN1 | DNM1 | (XIRP2) | PPP1R14D | TPM3 |
| ADGRV1 | DPF3 | LOXHD1 | PRPS1 | TPRN |
| AGR3 | DRGX | MAP1A | PTPRQ | TRIOBP |
| AK1 | EFCAB6 | MAPK10 | PTPRT | TTLL12 |
| AKAP5 | EFR3A | MCOLN3 | PTPRZ1 | TUBA3E |
| ANKRD24 | ELMOD1 | MPRIP | RAB26 | TUBAL3 |
| APPL2 | EML1 | MSN | RDX | TUBB2B |
| ARF1 | EPS8L2 | MSRB3 | RFX8 | TUBB6 |
| ARF4 | ESPN | MYH9 | RPS6KA2 | TWF2 |
| ARHGAP17 | ESPNL | MYO15A | RPS6KA5 | USH1C |
| ARMC4 | EZR | MYO1C | RSPH1 | USH1G |
| ATOH1 | FOXJ1 | MYO1H | RSPH9 | USH2A |
| ATP2B1 | FSCN1 | MYO3A | SCG3 | |
| ATP2B2 | FSCN2 | MYO3B | SERPINB6 | |
| ATP2B4 | GALNT9 | MYO6 | SGCB | |
| ATP6V1B2 | GAPDH | MYO7A | SGCG | |
| ATP6V1E1 | GDI2 | NFATC1 | SGIP1 | |
| ATP8B1 | GNAI1 | NMNAT2 | SH3GLB2 | |
| B3GNTL1 | GNAI2 | NPEPPS | SKOR2 | |
| BAIAP2L2 | GNAI3 | OCM | SLC17A8 | |
| BRSK2 | GNAL | OSBP2 | SLC8A1 | |
| CAB39L | GNAS | OSBPL11 | SLC9A3R2 | |
| CACNA2D2 | GNG4 | OSBPL1A | SMPX | |
| CALB2 | GPSM2 | OTOA | SPAG1 | |
| CAPZA1 | GPX2 | OTOF | SPTAN1 | |
| CAPZA2 | GRXCR1 | PAICS | STARD10 | |
| CAPZB | GRXCR2 | PAK1 | STXBP1 | |
| CCDC50 | GSTO1 | PAK2 | SYN3 | |
| CDH23 | HSF5 | PAK3 | TECTA | |
| CHRNA10 | HYDIN | PCDH15 | TECTB | |
| CHRNA9 | IRX2 | PDCD6IP | TMC1 | |
| CIB2 | KIAA1211L | PDK4 | TMC2 | |
| CKB | (KIAA1211) | PDZD7 | TMC5 | |
| CLIC5 | KIAA1549 | PGM2L1 | TMCC2 | |
| CORO2B | KIF1A | PHF21B | TMEM117 | |
| CRABP1 | KLHDC7A | PI4KA | TMEM255B | |
| CSNK2A1 | LCP1 | PITPNA | TMEM30A | |
| CTH | LHFPL5 | PITPNB | TMIE | |
| CUL1 | LHX3 | PLS1 | TMPRSS3 | |
| DFNB31 | LOC416212 | PLS3 | TMPRSS7 | |
| ABCA5 | (WHRN) | (NSG2) | PLSCR5 | TMSB4X |
| ACO1 | DFNB59 | LOC423919 | PNPT1 | TNNC2 |
| ACTB | (PJVK) | (SHTN1) | PODXL2 | TOLLIP |
| ACTG1 | DIAPH1 | LOC772075 | POU4F3 | TPM1 |
| ACTN1 | DNM1 | (XIRP2) | PPP1R14D | TPM3 |
| ADGRV1 | DPF3 | LOXHD1 | PRPS1 | TPRN |
| AGR3 | DRGX | MAP1A | PTPRQ | TRIOBP |
| AK1 | EFCAB6 | MAPK10 | PTPRT | TTLL12 |
| AKAP5 | EFR3A | MCOLN3 | PTPRZ1 | TUBA3E |
| ANKRD24 | ELMOD1 | MPRIP | RAB26 | TUBAL3 |
| APPL2 | EML1 | MSN | RDX | TUBB2B |
| ARF1 | EPS8L2 | MSRB3 | RFX8 | TUBB6 |
| ARF4 | ESPN | MYH9 | RPS6KA2 | TWF2 |
| ARHGAP17 | ESPNL | MYO15A | RPS6KA5 | USH1C |
| ARMC4 | EZR | MYO1C | RSPH1 | USH1G |
| ATOH1 | FOXJ1 | MYO1H | RSPH9 | USH2A |
| ATP2B1 | FSCN1 | MYO3A | SCG3 | |
| ATP2B2 | FSCN2 | MYO3B | SERPINB6 | |
| ATP2B4 | GALNT9 | MYO6 | SGCB | |
| ATP6V1B2 | GAPDH | MYO7A | SGCG | |
| ATP6V1E1 | GDI2 | NFATC1 | SGIP1 | |
| ATP8B1 | GNAI1 | NMNAT2 | SH3GLB2 | |
| B3GNTL1 | GNAI2 | NPEPPS | SKOR2 | |
| BAIAP2L2 | GNAI3 | OCM | SLC17A8 | |
| BRSK2 | GNAL | OSBP2 | SLC8A1 | |
| CAB39L | GNAS | OSBPL11 | SLC9A3R2 | |
| CACNA2D2 | GNG4 | OSBPL1A | SMPX | |
| CALB2 | GPSM2 | OTOA | SPAG1 | |
| CAPZA1 | GPX2 | OTOF | SPTAN1 | |
| CAPZA2 | GRXCR1 | PAICS | STARD10 | |
| CAPZB | GRXCR2 | PAK1 | STXBP1 | |
| CCDC50 | GSTO1 | PAK2 | SYN3 | |
| CDH23 | HSF5 | PAK3 | TECTA | |
| CHRNA10 | HYDIN | PCDH15 | TECTB | |
| CHRNA9 | IRX2 | PDCD6IP | TMC1 | |
| CIB2 | KIAA1211L | PDK4 | TMC2 | |
| CKB | (KIAA1211) | PDZD7 | TMC5 | |
| CLIC5 | KIAA1549 | PGM2L1 | TMCC2 | |
| CORO2B | KIF1A | PHF21B | TMEM117 | |
| CRABP1 | KLHDC7A | PI4KA | TMEM255B | |
| CSNK2A1 | LCP1 | PITPNA | TMEM30A | |
| CTH | LHFPL5 | PITPNB | TMIE | |
| CUL1 | LHX3 | PLS1 | TMPRSS3 | |
| DFNB31 | LOC416212 | PLS3 | TMPRSS7 |
Additional files
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Reporting standard 1
Reporting guidelines for mass spectrometry.
- https://doi.org/10.7554/eLife.50777.023
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Transparent reporting form
- https://doi.org/10.7554/eLife.50777.024
