Article Figures & Data
Figures
- Figure 1.
Differentially expressed gene clusters analyzed by SOMs. Differentially expressed genes were clustered into 36 centroids by SOMs (Partek). Each x-axis indicates individual time points. The y-axis shows the average normalized fold change (log2) for all the genes in the centroid, with the bars showing the range. The number within each centroid refers to a gene cluster referred to in the text. The specific number of genes and gene IDs in each cluster are listed in supplemental Table 4. Some of the correlations between DAVID functional annotations and patterns are shown within each centroid, with the following codes: AAB, amino acid biosynthesis; AAM, amino acid metabolism; AP, apoptosis; CA, channel activity; CB, calcium binding; CC, cell cycle; CM, cell migration; CYT, cytoskeleton; DD, DNA damage; EM, extracellular matrix; ER, endoplasmic reticulum; GLY, glycosylation; HLH, helix loop helix genes; IFN, interferon responses; MIB, metal ion binding; MP, membrane proteins; NB, nucleolus biosynthesis; ND, neural development; NOS, nuclear organelle synthesis; PAT, patterning; RB, RNA binding; RD, regulation of differentiation; SPS, sensory perception of sound; SR, stress response; TM, transmembrane transport; and VGC, voltage gated channels.
- Figure 2.
Phenotypic measurements across the regenerative time course. A, DNA replication in supporting cells was measured by a BrdU incorporation assay. The x-axis indicates individual time points. The y-axis shows the mean number of BrdU-labeled cells per 10,000 μm2 ± SD in control and streptomycin-treated organotypic cultures. B, The number of mature hair cells was quantified by measuring MYO7a-labeled cells. Chicken utricle sensory epithelia organotypic cultures were treated with streptomycin and immunohistochemically labeled for MYO7a to detect mature hair cells. The x-axis indicates individual time points. The y-axis shows the mean number of hair cells per 10,000 μm2 ± SD.
- Figure 3.
Expression profiles of 32 known hair cell markers. The x-axis indicates individual time points. The y-axis shows the fold change of streptomycin-treated versus control samples on a log2 scale. These sentinel genes are significantly downregulated at the 0 and 24 h time points and showed a recovery trend toward the end of the time course.
- Figure 4.
SOMs of potential hair cell markers. Genes that passed the filters for potential hair cell markers (downregulated >2-fold at 0–24 h and a recovery trend by the 168 h time point) were clustered into 25 groups by SOMs (Partek). The x-axis of each group indicates individual time points. The y-axis shows the normalized fold change.
- Figure 5.
Immunohistochemical staining of novel hair cell markers. Chicken utricles were labeled with antibodies to known hair markers (MYO7a or otoferlin) and to potential novel hair cell markers identified in the present study. Scale bar, 16 μm. A, DNM1 (green) and MYO7a (red) are colocalized within all hair cells and are expressed at similar levels. B, Hair cells are labeled for otoferlin (green). AP3B2 (red) is expressed homogeneously within the cytoplasm of hair cells. C, MYO7a (green) labels hair cells and MDFI (red) labels the nuclei of a subpopulation of hair cells at various intensity levels. The images show the apical surface of the sensory epithelium with supporting cells filling the gaps between the labeled hair cells.
- Figure 6.
Examples of gene networks within the clustered data. A, Network of interactions between putative hair-cell-specific genes. All 526 hair-cell-specific gene names from the clustering analysis were uploaded to g:Profiler and analyzed as described in the Materials and Methods. This figure includes only genes detectably expressed in the regenerative time course. Red circles denote genes within the 526 putative hair-cell-specific input set. Red lines indicate interactions between those genes/gene products. Blue circles denote additional genes within the differentially expressed subset. Black circles denote genes that are detectable but not differentially expressed. Black lines denote interactions between genes/gene products. B, Network of interactions between some of the transcription factor genes within clusters 13 and 15 (Fig. 8) exhibiting a dip in expression at 120 h. This includes only genes that are detectably expressed in the regenerative time course. Color coding as in A except red genes are differentially expressed transcription factors in these clusters.
- Figure 7.
Changes in specific components of the NOTCH and FGF signaling pathways during regeneration. The x-axis indicates individual time points. The y-axis shows the fold change of streptomycin-treated versus control samples on a log2 scale. A, Expression profiles of ATOH1, DLL1, and HES5 at 24 h intervals across the 168 h time course. B, Expression profiles of HES5 and HES7 with the addition of detailed sampling within the 48–72 h period at 6 h intervals. C, Expression profiles of ATOH1, HES6, and HEYL. D, Expression profiles of differentially expressed intracrine FGFs (FGF12 and FGF14). E, Expression profiles of differentially expressed paracrine FGFs (FGF3, FGF16, and FGF20). F, Expression profiles of FGF19, FGFR1, and FGFR3.
- Figure 8.
Gene expression profiles of differentially expressed transcription factors clustered into 16 centroids by SOMs. The 212 differentially expressed transcription factors were grouped by SOMs (Partek) according to their expression patterns. The x-axis of each group indicates individual time points. The y-axis shows the normalized fold change on a log2 scale. Centroids were then manually grouped according to their shared overall expression patterns. See Differentially expressed transcription factor genes in the Results for a description of patterns A–J.
Tables
Gene Description BSN Bassoon (presynaptic cytomatrix protein) CAB39L Calcium binding protein 39-like CACNA1B Calcium channel, voltage-dependent, N type, alpha 1B subunit CADM2 Cell adhesion molecule 2 CAPSL Calcyphosine-like CASQ2 Calsequestrin 2 (cardiac muscle) GPR143 G protein-coupled receptor 143 GPR149 G protein-coupled receptor 149 GPR98 G protein-coupled receptor 98 KCNA1 Potassium voltage-gated channel, shaker-related subfamily, member 1 KCNA3 Potassium voltage-gated channel, shaker-related subfamily, member 3 KCNA4 Potassium voltage-gated channel, shaker-related subfamily, member 4 KCNAB1 Potassium voltage-gated channel, shaker-related subfamily, beta member 1 KCNAB2 Potassium voltage-gated channel, shaker-related subfamily, beta member 2 KCNH6 Potassium voltage-gated channel, subfamily H (eag-related), member 6 KCNJ2 Potassium inwardly-rectifying channel, subfamily J, member 2 KCNS2 Potassium voltage-gated channel, delayed-rectifier, subfamily S, member 2 KCNU1 Potassium channel, subfamily U, member 1 MYBPC1 Myosin binding protein C, slow type MYH7B Myosin, heavy chain 7B, cardiac muscle, beta MYLK4 Myosin light chain kinase family, member 4 MYO18B Myosin XVIIIB MYO1H Myosin IH MYO3A Myosin IIIA MYO3B Myosin IIIB PCDH20 Protocadherin 20 PCDH8 Protocadherin 8 PCDH9 Protocadherin 9 PCLO Piccolo (presynaptic cytomatrix protein) PCP4 Purkinje cell protein 4 SLITRK4 SLIT and NTRK-like family, member 4 SMPX Small muscle protein, X-linked SNTN Sentan, cilia apical structure protein SPHKAP SPHK1 interactor, AKAP domain containing SPINK4 Serine peptidase inhibitor, Kazal type 4 SPOCK3 Sparc/osteonectin, cwcv and kazal-like domains proteoglycan SPRY3 Sprouty homolog 3 (Drosophila) SV2B Synaptic vesicle glycoprotein 2B SYN3 Synapsin III SYNC Syncoilin, intermediate filament protein SYT10 Synaptotagmin X TESC Tescalcin TM4SF18 Transmembrane 4 L six family member 18 TMC5 Transmembrane channel-like 5 TMCC2 Transmembrane and coiled-coil domain family 2 TMEM117 Transmembrane protein 117 TMEM151B Transmembrane protein 151B TMIGD1 Transmembrane and immunoglobulin domain containing 1 TMPRSS7 Transmembrane protease, serine 7 TMPRSS9 Transmembrane protease, serine 9 Fifty examples of putative new hair-cell-specific markers are listed from the overall list of 494 genes (available online) identified by our pattern and cluster analysis.
Description Gene Hair-cell-specific transcription factors Ankyrin repeat domain 5 ANKRD5 D4, zinc and double PHD fingers, family 3 DPF3 Dorsal root ganglia homeobox DRGX Forkhead box F1 FOXF1 Growth factor independent 1 transcription repressor GFI1a Iroquois homeobox 2 IRX2 LIM homeobox 3 LHX3a Myod family inhibitor MDFIb MLX interacting protein-like MLXIPL Nuclear factor of activated T-cells, cytoplasmic, calcineurin-dependent 1 NFATC1 Brain-specific homeobox/POU domain protein 3 POU4F3a SKI family transcriptional corepressor 1 SKOR1 SKI family transcriptional corepressor 2 SKOR2 Zinc finger protein 385B ZNF385B Transcription factor gene expression clusters Dip at 54 hours (18 genes) coincident with phenotypic conversion (centroid number = 7) B-cell translocation gene 1, anti-proliferative BTG1 Cbp/p300-interacting transactivator, with Glu/Asp-rich carboxy-terminal domain, 4 CITED4 Hairy and enhancer of split 5 HES5 Hairy and enhancer of split 7 HES7 Interferon regulatory factor 4 IRF4 Hairy and enhancer of split 5-like LOC419390 Myod family inhibitor MDFI PR domain containing 5 PRDM5 Prickle homolog 1 PRICKLE1 Prickle homolog 2 PRICKLE2 Synovial sarcoma translocation gene on chromosome 18-like 1 SS18L1 Storkhead box 1 STOX1 Tripartite motif containing 24 TRIM24 Teashirt zinc finger homeobox 1 TSHZ1 Zinc finger, matrin-type 4 ZMAT4 Zinc finger protein 385B ZNF385B Zinc finger protein 395 ZNF395 Zinc finger protein 804A ZNF804A Dip at 120 hours (25 genes) coincident with cell cycle cessation (centroid number = 13) AT rich interactive domain 5A (MRF1-like) ARID5A Camp responsive element modulator CREM Histone deacetylase 7 HDAC7 Inhibitor of DNA binding 2, dominant negative helix-loop-helix protein ID2 Notch 1 NOTCH1 Nuclear receptor subfamily 4, group A, member 3 NR4A3 Peroxisome proliferator-activated receptor delta PPARD Serum response factor (c-fos serum response element-binding transcription factor) SRF Tripartite motif containing 45 TRIM45 Zinc finger protein 503 ZNF503 Zinc finger protein 541 ZNF541 Atonal homolog 1 ATOH1 Distal-less homeobox 5 DLX5 Distal-less homeobox 6 DLX6 Ets homologous factor EHF E74-like factor 3 (ets domain transcription factor, epithelial-specific) ELF3 Forkhead box J1 FOXJ1 Hematopoietic cell-specific Lyn substrate 1 HSLS1 Hairy and enhancer of split 6 HES6 Hairy/enhancer-of-split related with YRPW motif-like HEYL Inhibitor of DNA binding 1, dominant negative helix-loop-helix protein ID1 Inhibitor of DNA binding 4, dominant negative helix-loop-helix protein ID4 MLX interacting protein-like MLXIPL SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily c, member 1 SMARCC1 Zinc finger, MYND-type containing 10 ZMYND10 Peaks at 54–72 hours (8 genes) coincident with phenotypic conversion (centroid number = 16) Ankyrin repeat domain 33 ANKRD33 Four and a half LIM domains 2 FHL2 Forkhead box F1 FOXF1 Iroquois homeobox 2 IRX2 LIM domain only 3 (rhombotin-like 2) LMO3 Mastermind-like domain containing 1 MAMLD1 Recombination signal binding protein for immunoglobulin kappa J region-like RBPJL Vitamin D (1,25-dihydroxyvitamin D3) receptor VDR There are 14 transcription factor genes that are potential new hair cell markers by clustering analysis. A list of transcription factor genes that correlate with three of the patterns of gene expression are shown in Figure 8. All gene lists and centroid numbers are available online.
↵aKnown hair cell markers.
↵bValidated in this study (see Fig. 5).
